Method Development and Validation for the Analysis of Coumarin-based Phototoxins in Citrus-derived Essential Oils Using Liquid Chromatography-Mass Spectrometry

Method Development and Validation for the Analysis of Coumarin-based Phototoxins in Citrus-derived Essential Oils Using Liquid Chromatography-Mass Spectrometry | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Method Development and Validation for the Analysis of Coumarin-based Phototoxins in Citrus-derived Essential Oils Using Liquid Chromatography-Mass Spectrometry Siheng Li, Colleen Kelly, Radim Knob, Kent McConnell, Justin Barrett This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3318708/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The occurrence of coumarin-based photoactive compounds in essential oil has been a concern due to the adverse health effects it has on human health. A fit-for-purpose analytical method for major coumarin-based phototoxins is critical to guarantee the essential oils product safety and compliance with the regulatory requirements. Herein, a liquid chromatography-mass spectrometry (LC-MS) analytical method was developed to identify and quantify 25 coumarin derivatives in essential oils. The sample preparation procedure was optimized for essential oils to use selective solvent extraction to remove the limonene matrix interference. To achieve the optimal sensitivity and specificity, UHPLC column of biphenyl stationary phase was used for this analysis. An inter-laboratory validation study was conducted to evaluate the method performance. The limit of quantification (LOQ) for all target analytes were validated at 5 µg/g. Satisfactory recoveries were obtained for the majority of analytes in the range of 80-115%. Both the repeatability RSDr (within-laboratory) and reproducibility RSDR (between-laboratory) <10%. This method was demonstrated as applicable for the quality control testing of coumarin-based phototoxins in essential oils. coumarins furocoumarins essential oils LC-MS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Coumarins belong to a family of naturally-occurring benzenoid lactone phytochemicals widely present in many plant species.(Pereira et al. 2018 ) They are secondary metabolites that typically are more concentrated in fruits and leaves, where they serve the purpose to expel insects and defend against fungal and bacterial infections. The variation of the functional groups during the plant biosynthesis results in the presence of numerous distinct coumarin-derivatives. The prominent sub-class of coumarin-derivatives is furocoumarins (also called furanocoumarins), which is defined by the core tricyclic structure of a furan ring unit fused onto a coumarin motif, such as angelicin, bergamottin, bergapten and psoralen.(Santana et al. 2004 ) Plant materials containing coumarin derivatives have been used in traditional health practices since ancient Egyptian times for a variety of benefits.(Khursheed and Jain 2021 ) However, the photoallergenicity and phototoxicity of certain coumarin derivatives were gradually recognized with the advance of toxicological research. Human exposure to certain coumarin derivatives in the presence of long wavelength ultraviolet light (320 to 380 nm) can result in an acute skin allergy that may persist for several months.(Tisserand and Young 2014b ) Furthermore, furocoumarins are also reported as potential photo-mutagen and photo-carcinogen.(Melough et al. 2018 ) Due to adverse health effects, the level of several coumarin derivatives in natural herbal products are strictly regulated in many countries. Currently, the European regulation (EC) No 1223/2009 prohibits the use of furocoumarins except for the normal content in natural essences.(European Commission 2009 ) In 2008, The International Fragrance Association (IFRA) added six furocoumarins (bergapten, bergamottin, byakangelicol, epoxybergamottin, isopimpinellin and oxypeucedanin) as regulated furocoumarin marker compounds, with their levels in finished cosmetics to no more than 5 ppm for leave-on products and 50 ppm for rinse off products.(IFRA 2008 ) In 2015, the updated IFRA standard set the limit of bergapten in consumer essential oil products as no more than 15 ppm.(IFRA 2015 ) The level of 6-methylcoumarin and 7-methoxycoumarin in fragrance products and cosmetics was also restricted by IFRA and the other major regulatory agencies.(Cui et al. 2021 ) With strong customer demand, the global essential oils market is projected to grow by 9.57% compound annual growth rate (CAGR) to 18.25 billion USD in 2028.(Fortune Business Insights 2021 ) The total amount of essential oils produced worldwide has already reach 300,000 metric tons, with more than 70% of the essential oils used in fragrance, flavor and aromatherapy sector. Citrus-derived essential oils constituted 40% market share in the global essential oils market due to their availability and ideal organoleptic property. However, citrus plants are known to contain many coumarin derivatives.(Russo et al. 2021 ) Citrus-derived essential oils are typically produced by cold pressing or steam distillation of the corresponding citrus plant materials. Thus, elevated levels of coumarin derivatives is not unusual in citrus oils, especially for essential oils produced by the cold-pressing of citrus peel, due to the facilitated co-extraction of the relatively polar and nonvolatile coumarins.(Tisserand and Young 2014a ) As secondary plant metabolites, the chemical profile of coumarin derivatives in herbal extracts is highly variable, which is dictated by genetic diversity and also by environmental factors such as growing season and geographical origin of the plants.(Frérot and Decorzant 2004 ) Considering the consumer essential oil products are commonly applied to the skin, it is critical to have a suitable analytical method for coumarin-based phototoxins in place as part of the quality control process to guarantee the essential oils are safe for consumers and meet regulatory safety requirements. Several furocoumarin analytical methods using various analytical techniques have been reported in the literature. They focus on a small subset of coumarin-derivatives in relatively simple sample matrices such as fruit and fruit juices.(Melough et al. 2017 ; Lin et al. 2009 ; Govindarajan et al. 2007 ; Kreidl et al. 2020 ; Prosen and Kocar 2008 ) Due to the need for high-throughput quality control (QC) testing of essential oils, it is desirable to have a more comprehensive analytical method capable of monitoring all coumarin derivatives with safety concerns. The sample matrix of essential oils is highly complex and composed of concentrated phytochemicals. Based on our initial investigation, many reported analytical methods lack satisfactory method performance for these sample matrices. A 15-analyte high performance liquid chromatography (HPLC) method adopted by IFRA was intended for furocoumarins analysis in essential oils, but a long HPLC run time and limited capability to resolve matrix interferences in complex essential oils by UV detection hinder its wide application.(Macmaster et al. 2012 ; Frérot and Decorzant 2004 ) Recently, other multi-analyte analytical methods have been reported but they have yet to be demonstrated with acceptable method reproducibility across laboratories.(Dugrand et al. 2013 ; Arigo et al. 2021 ) Herein, we report the development of a multiplexed liquid chromatography-mass spectrometry (LC-MS) method to analyze 25 coumarin derivatives with known or potential safety concerns.(Melough and Chun 2018 ; Melough et al. 2018 ) The method development was mainly focused on the optimization of instrumental conditions and sample preparation to facilitate reliable analysis in the challenging matrix of essential oils. An inter-laboratory validation study was conducted in two laboratories in accordance with International Council of Harmonisation (ICH) guidelines on validation of analytical procedures and the methodology to demonstrate acceptable method performance in the essential oil matrix. Materials and Methods Reagents and materials Methanol (LCMS grade), hexane (HPLC grade) and formic acid (LCMS grade) were obtained from Millipore Sigma. Discovery ® DSC-18 SPE bulk packing was purchased from Sigma-Aldrich. A 1 mg/mL standard solution containing 15 furocoumarins (bergamottin, bergapten, byakangelicin, byakangelicol, epoxybergamottin, heraclenin, imperatorin, isoimperatorin, isopimpinellin, oxypeucedanin, oxypeucedanin hydrate, phellopterin, psoralen, 8-geranyloxypsoralen, 8-MOP) in acetonitrile was supplied by Chromadex (Irvine, CA, USA). The reference materials of citropten, 6-methylcoumarin, 7-methoxycoumarin and trioxalen were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bergaptol (> 97%) was obtained from Toronto Research Chemicals (Toronto, ON, Canada). The reference materials of 5-geranoxy-7-methoxycoumarin, angelicin, isobergapten, pimpinellin and sphondin were obtained from TargetMOL (Wellesley Hills, MA, USA). Standard Preparation The stock standard solution of each individual analyte was prepared at 800–2000 µg/mL in appropriate solvent (methanol or ethanol) based on their solubility. A composite solution containing all 25 analytes at 100 µg/mL was prepared by mixing appropriate volumes of the stock standard solutions. The composite solution was further diluted to yield an intermediate stock solution at 20 µg/mL. The stock solution and intermediate stock solution were stored frozen for up to 3 months. Calibration standards containing each analyte at 0.04, 0.08, 0.20, 0.40 and 1.00 µg/mL were prepared by diluting the intermediate stock solution in 70/30 methanol/water (v/v). Sample Preparation The essential oil samples used in this study were obtained from in-house production. A 1 g sample was weighed into a 10 mL volumetric flask and extracted with 10 mL 0.01% formic acid in 90/10 methanol/water (v/v), with 5 minutes sonication. The sample extract was transferred into a polypropylene centrifuge tube and centrifuged at 3000 × g for 5 minutes. 1 mL aliquot of the top layer was transferred into a microcentrifuge tube and extracted with 50 µL hexane. After thorough mixing, the sample tube was centrifuged at 12000 × g at 4°C for 5 minutes and the top hexane layer was discarded. The sample extract was transferred into a new microcentrifuge tube containing 50 mg DSC-18 SPE sorbent. After incubation at room temperature for 10 minutes, the sample extract was filtered (0.2 µm PTFE filter, Phenomenex) and an 80 µL aliquot was mixed with 920 µL 70/30 methanol/water (v/v) in an autosampler vial for LC-MS analysis. LC-MS analysis LC-MS analyses were performed on an Agilent 1290 Infinity LC system coupled with an Agilent 6125 MSD single-quadrupole mass spectrometer equipped with electrospray ionization (ESI) source. Data acquisition and processing were controlled by Agilent OpenLab software. Chromatographic separation was performed using a Phenomenex Kinetex ® biphenyl column (100 × 2.1 mm, 1.7 µm, 100 A). Mobile phase A was 0.01% formic acid in water and mobile phase B was methanol (LCMS Grade). The gradient elution program was as follows: 0–4.0 min 50–60%B, 4.0–11.0 min 60–95%B, 11.0–12.0 min 95%B, 12.0-12.1 min 95 − 50%B, 12.1–14.0 min 50%B. LC flow rate was 0.4 mL/min. The column oven was set to 40°C and the autosampler was at 5°C. The injection volume was 2 µL. The MS acquisition was carried out in positive ESI mode to monitor the SIMs in Table 1 . The following MS source settings were applied: 340°C gas temperature, 11 L/min gas flow, 32 psi nebulizer gas, 4000 V capillary voltage. Table 1 The targeted coumarin derivatives, their partition coefficient (LogP) and their specific MS parameters for the identification and quantitation. Analyte CAS Mw LogP a SIM 1 Fragmenter (V) SIM 2 Fragmenter (V) 6-Methylcoumarin 92-48-8 160.2 2.3 161.0 120 105.0 220 7-Methoxycoumarin 531-59-9 176.2 1.6 177.0 150 121.0 200 Psoralen 66-97-7 186.2 1.9 187.0 130 131.0 210 Angelicin 523-50-2 186.2 1.9 187.0 130 131.0 210 Bergaptol 486-60-2 202.2 1.6 203.0 120 147.0 210 Citropten 487-06-9 206.2 1.5 207.0 140 192.0 200 Methoxsalen (8-MOP) 298-81-7 216.2 1.8 202.0 210 174.0 230 Sphondin 483-66-9 216.2 1.8 202.0 210 174.0 230 Bergapten 484-20-8 216.2 1.8 202.0 210 174.0 230 Isobergapten 482-48-4 216.2 1.8 202.0 210 174.0 230 Trioxsalen 3902-71-4 228.2 3.0 229.0 140 115.0 260 Isopimpinellin 482-27-9 246.2 1.6 247.0 140 232.0 190 Pimpinellin 131-12-4 246.2 1.6 247.0 140 232.0 190 Imperatorin 482-44-0 270.3 3.1 271.0 80 203.0 160 Isoimperatorin 482-45-1 270.3 3.1 271.0 80 203.0 160 Oxyimperatorin 35740-18-2 286.3 2.3 287.1 130 203.0 190 Oxypeucedanin 3173-02-2 286.3 2.3 287.1 130 203.0 190 Phellopterin 2543-94-4 300.3 3.0 218.0 220 301.0 90 Oxypeucednin hydrate 2643-85-8 304.3 1.2 305.1 150 147.0 260 Byakangelicol 26091-79-2 316.3 2.2 231.0 180 317.1 130 5-Geranoxy-7-methoxycoumarin 7380-39-4 328.4 4.5 193.0 190 329.1 90 Byakangelicin 482-25-7 334.3 1.0 317.1 130 231.0 180 8-Geranyloxypsoralen 7437-55-0 338.4 4.8 203.0 160 339.1 80 Bergamottin 7380-40-7 338.4 4.8 203.0 160 339.1 80 Epoxybergamottin 206978-14-5 354.4 3.8 337.1 90 355.2 70 a The partition coefficients (LogP) were obtained from Chemicalize database Method Validation The optimized analytical method was validated in accordance with ICH guidelines, including the evaluation of specificity, linearity, limit of quantitation (LOQ), accuracy, and precision.(International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use 2005 ) Yuzu oil was selected as a representative citrus-derived oil matrix because it was determined to be an analyte-free matrix. The linearity was determined by the correlation coefficients of the calibration curves. Specificity and LOQ were evaluated by the fortification of yuzu oil with each analyte at the lowest level to achieve the acceptable method performance. Accuracy and repeatability were evaluated by spiking yuzu oil with each analyte at four concentration levels (5, 20, 40 and 48 µg/g) in 3 to 6 replicates. The quantitation was performed using solvent calibration standards. The validation experiment was conducted in two doTERRA laboratories to evaluate reproducibility. Results and Discussion Optimization of Chromatographic and MS Conditions All 25 target analytes presented in Table 1 were selected based on current global regulatory requirements and potential safety concerns. In consideration of the low targeted analytical range, the complexity of essential oil sample matrix, and the necessity to differentiate each coumarin derivative, LC-MS was considered as the most appropriate analytical technique. Although LC-tandem mass spectrometry (LC-MS/MS) and LC-high resolution mass spectrometry (LC-HRMS) offer the most ideal selectivity and sensitivity, a properly developed LC-MS method in single ion monitoring (SIM) mode would provide sufficient method performance for this analysis. Furthermore, it is relatively easy to operate and maintain a single-quadrupole mass spectrometer and harmonize this method in multiple laboratories for quality control testing. Suitable chromatographic conditions are essential to achieve optimal method specificity and sensitivity by minimizing matrix interference and resolving critical pairs of target analytes. In general, reversed-phase chromatography is amenable to the reasonable retention of coumarin derivatives. However, it is a challenge to accommodate 25 targeted coumarin derivatives with acceptable selectivity in a short LC analysis. In particular, the target analytes included six groups of isobaric analytes that are not feasible to be resolved by the MS detector, hence their selective identification fully relied on the resolving power of LC separation. To determine the most ideal analytical column for this method, the separation of the target analytes using UHPLC columns of different stationary phases was investigated. As shown in Fig. 1 , the chromatograms were obtained from the analyses on polar C18, phenyl-hexyl, fluoro-phenyl and biphenyl columns of the same dimensions (100 × 2.1 mm) using identical LC gradients of water and methanol. The biphenyl column clearly offered the best retention for all the target analytes, attributed to the strong pi-pi interaction between the coumarin motif and the biphenyl stationary phase. Fewer analytes coeluted using the biphenyl column, with only three groups of coeluting clusters (6-methylcoumarin and psoralen; oxyimperatorin, isobergapten and pimpinellin; isoimperatorin and phellopterin) which can be resolved by MS detector based on their m/z difference of molecular ions. In comparison, the coelution of 8 to 14 analytes were observed using the other three types of columns. With a slight adjustment to a shallower LC gradient, baseline separation of all the critical analyte pairs was achieved, including the group of four isobaric analytes 8-MOP, sphondin, bergapten and isobergapten. Therefore, the biphenyl column was chosen to perform all the subsequent optimizations. The total analysis time of one injection was 14 minutes, which significantly improves testing throughput compared to many published methods.(Govindarajan et al. 2007 ; Lin et al. 2009 ; Macmaster et al. 2012 ; Dugrand et al. 2013 ) However, it was noted that bergaptol was unable to be resolved from its isobaric isomer xanthotoxol on the biphenyl column. Bergaptol and xanthotoxol can either be quantified as a sum or will require an alternative LC separation approach. A water-methanol mixture was used as eluent due to the incompatibility of acetonitrile with biphenyl stationary phase.(Yang et al. 2005 ) To improve peak shape and facilitate the ionization, modifications to the mobile phase pH were investigated. The peak shape and retention time remained constant with the increase of mobile phase acidity, attributed to the lack of ionizable functional groups in the targeted coumarin-derivatives. (Fig. 2 ) However, it is noteworthy that the increased acid concentration actually suppressed the ionization process, resulting in lower MS response of certain analytes. In particular, the peak area of 7-methoxycoumarin was 20% higher in pH 3.5 compared to pH 2.7. Taking this into consideration, the addition of 0.01% formic acid as the additive of mobile phase A was sufficient for improved peak shape and optimal MS response. All of the target coumarin derivatives yielded stable protonated molecular ions [M + H] + in positive electrospray ionization (ESI). Most of the molecular ions are well suited in the selected ion monitoring (SIM) to represent each target analyte. However, several molecular ions were less specific due to the high MS background and the susceptibility to matrix interference, including bergapten, sphondin, 8-MOP and isobergapten (m/z 217.0); phellopterin (m/z 301.0); byakangelicin (m/z 335.1); and epoxybergapmottin (m/z 355.2). (Fig. 3 ) In order to find the most suitable ions in SIM acquisition to represent each of the target analytes, the fragmentor voltage was ramped up in 10 V increments from 70 V to 250 V to encourage collision-induced dissociation of the molecular ions. Through this process, we were able to identify the most specific signature fragment ions of each target analyte and their respective optimal fragmentor voltage. In particular, fragment ions of bergapten, sphondin, 8-MOP and isobergapten (m/z 202.0); phellopterin (m/z 218.0); byakangelicin (m/z 317.1); and epoxybergapmottin (m/z 337.1) were identified to offer significantly improved signal-to-noise ratio (S/N) compared to their molecular ions. The top two ions of each target analyte were selected as quantifier ion and qualifier ion in the SIM acquisition, which further improved the method specificity. The complete list of the SIM ions and fragmentor voltages are summarized in Table 1 . The optimized chromatography and MS conditions enabled sensitive detection of most target analytes down to 10 to 20 pg on column. 7-methoxycoumarin and epoxybergamottin were the least sensitive analytes due to the higher background of their respective SIM ions. Overall, 40 pg on column was an achievable limit of detection (LOD) for all the target analytes with corresponding S/N more than 3. Development of Sample Preparation Procedure The major constituents of citrus-derived essential oils are various monoterpenes, such as limonene, α-pinene, γ-terpinene and myrcene, with limonene as the most abundant constituent commonly above 50% (w/w).(Kvittingen et al. 2021 ) Although monoterpenes are hydrocarbons that are not ionizable by ESI, they share elution profiles similar to coumarin derivatives in reversed-phase chromatography. To understand whether the analysis of coumarin-derivatives is affected by the presence of concentrated limonene in the citrus-derived oil, distilled wild orange oil fortified with the target analytes was dissolved in methanol and analyzed by a LC-UV-MS system, with UV detection at the apex of limonene UV-Vis spectrum (206 nm). Although most of the target analytes were not affected, suppressed response and retention time shifts were observed in the elution of imperatorin (8.5 min), trioxsalen (8.6 min) and isoimperatorin (9.3 min), which happened to overlap with the elution region of limonene (8.3–8.8 min) (Fig. 4AI-AIII). Column performance and MS ionization were compromised by the overloading of an abundance of limonene in the tested sample. Thus, additional sample clean-up to remove limonene is required for the accurate analysis of all the targeted coumarin-derivatives. QuEChERS is the most widely used sample preparation in food and environmental applications due to excellent cost-effectiveness.(Anastassiades et al. 2003 ; Majors et al. 2010 ) It is the primary solution to extract organic residues from many food matrices, such as protein, carbohydrates and lipids. However, it is ineffective at selectively extracting target analytes from small molecule matrix components with similar partition coefficients. The calculated partition coefficient of limonene (logP = 3.22) is within the range of the partition coefficients of the target analytes (logP 1.0–4.8), indicating a tendency of QuEChERS to co-extract both the analytes and limonene into the acetonitrile phase (Table 1 ). Thus, an alternative technique to remove the monoterpene matrix was explored for this analysis. The use of an appropriate, selective extraction solvent turned out to be a more suitable sample preparation technique. Although limonene is soluble in medium polarity solvents such as methanol and acetonitrile, the use of water as a co-solvent effectively minimizes its solubility. On the other hand, coumarin derivatives are relatively more soluble in a methanol-water mixture. A water-methanol mixture as the extraction solvent is capable of extracting coumarin derivatives in the presence of monoterpene sample matrix. 10% water in methanol (v/v) was determined as the optimal ratio in removing abundant limonene while maintaining acceptable recovery of all target analytes. After centrifugation, most of the sample matrix precipitated while the target analytes extracted into the upper layer of the extraction solvent. Further increases of water ratio marginally improved the removal of limonene but resulted in much higher recovery loss of the least polar analytes, such as bergamottin and 5-geranoxy-7-methoxycoumarin. For a sample containing > 90% limonene, 10% water in methanol (v/v) as the extraction solvent is not sufficient to remove all the matrix interference for LC-MS analysis (Fig. 4BI-III). To further improve limonene removal, an additional step of liquid-liquid extraction with hexane was introduced. A sample diluted with 10% methanol was mixed with hexane in a 1:20 ratio, then it was partitioned by centrifugation and the hexane layer was discarded. This enabled the removal of the matrix interference associated with residual limonene in the sample extract. As shown in Fig. 4III, the limonene level in the wild orange oil sample extract after liquid-liquid extraction was significantly reduced compared to the methanol/water extract. The retention time of trioxsalen, imperatorin and isoimperatorin in the wild orange oil sample extract also overlapped well with a solvent standard of the same concentration, indicating the successful removal of limonene interference (Fig. 4CI-II). Subsequently, the sample was incubated with C18-based dispersive solid phase extraction (dSPE) sorbent to remove residual hexane that can cause retention time shifts. The optimized extraction procedure was evaluated through the spiked recovery of target analytes in various essential oils at 5 µg/g, which is the intended LOQ of this analysis. Nineteen essential oils were screened as representative of herbal extracts derived from various citrus peels, leaves and flowers, as well as from the similar botanical family of Rutaceae and Apiaceae with reported phototoxicity. In general, the spiked recoveries were mostly within the acceptable range (75–120%), as represented by the green color in Fig. 5 . Several essential oils produced from citrus peels contain measurable levels of targeted coumarin-derivatives that hinder spiked recovery determination at 5 µg/g fortification level. Thus, their recoveries were not determined (ND). Out of these 25 targeted compounds, epoxybergamottin, bergamottin, 8-geranyloxypsoralen and 5-geranoxy-7-methoxycoumarin showed lower spiked recovery. The high lipophilicity of epoxybergamottin (LogP = 3.8), bergamottin (LogP = 4.8), 8-geranyloxypsoralen (logP = 4.8) and 5-geranoxy-7-methoxycoumarin (LogP = 4.5) was presumably causing their recovery loss during the sample extraction. Overall, this optimized sample extraction procedure was demonstrated to be applicable for various types of essential oils. Method Validation An inter-laboratory method validation study was conducted in accordance with ICH guidelines on validation of analytical procedures.(International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use 2005 ) Based on ICH guidelines, this method is considered as a category II Quantitative Impurity Assay, thus the method performance characteristics evaluated as part of the validation study included specificity, linearity, range, limit of quantitation (LOQ), accuracy and precision. The method specificity was investigated by comparing a blank sample with the same blank sample spiked with the target analytes. The selection of a blank citrus essential oil free of coumarin derivatives was rather limited because of naturally occurring coumarin derivatives inherent in most of citrus oils. Through our initial sample screening, yuzu oil was chosen as a representative blank sample without detectable amounts of target analytes. All the primary SIMs were shown to be highly selective with no chromatographic interference observed in yuzu oil. The chromatograms are presented in supporting information (Figure S1 ). The secondary SIMs were less selective and used as qualifier ions for confirmation purpose. Overall, successful analyte identification was demonstrated at concentrations equal to or above the method LOQ. The analytical range evaluated in this study was from 0.04 µg/mL to 1 µg/mL, which was equivalent to the range of 5 µg/g to 125 µg/g in the essential oil sample. Calibration curves were established by a set of five calibration standards using quadratic regression and 1/x weighting. The quadratic standard curves provided a better fit due to a non-linear response near the upper end of the calibration curves, presumably related to saturation during ionization.(Yuan et al. 2012 ) Over the course of the validation study, the correlation coefficient (r) of all target analytes were consistently above 0.99, which demonstrates acceptable linearity of this method (Table S1 ). Due to the absence of citrus-derived oils with certified levels of coumarin derivatives, the method accuracy was evaluated by the marginal recovery% of the blank sample fortified with target analytes at four concentration levels (5 µg/g, 20 µg/g, 40 µg/g and 48 µg/g). Repeatability (RSD r ) was determined from the analysis of the fortified sample in three to six replicates, and the inter-laboratory reproducibility (RSD R ) was determined from the repeatability results from two independent laboratories. As shown in Fig. 6 A and Table S2, acceptable recoveries and RSDs were obtained in the validation study. The mean recoveries were within 80–115%, except bergamottin and 5-geranoxy-7-methoxycoumarin. The lower mean recoveries in the range of 60–80% were presumably due to recovery loss during the extraction because of their high lipophilicity. The RSD r of all the analytes at 5 µg/g fortification level was between 4–10%. In higher fortification levels, the analyses were more precise with RSD r of all 25 targeted analytes below 5%. The precision results obtained from two laboratories was also aligned with the single-laboratory results, with RSD R below 6% for all the analytes (Fig. 6 B). The narrow distribution of RSD r and RSD R demonstrates the high consistency and precision of this analytical method. The LOQs were determined as the lowest fortification level that met the identification criteria and showed acceptable recovery and precision. Yuzu oil fortified at 5 µg/g had a S/N consistently above 10 for all 25 analytes in the two laboratories, as shown in Table S3. The spike recovery and precision also met their respective acceptance criteria. Therefore, the LOQ of this analytical method is set as 5 µg/g. Conclusion A multiplexed LC-MS method was developed and thoroughly validated for the analysis of coumarin-derivatives in essential oils. This method enables the analysis of many important coumarin-based phototoxins in essential oils by using simple sample preparation and easy to operate instrumentation. An optimized extraction solvent was used to extract the coumarin-derivatives and remove the limonene matrix interference simultaneously. The method was demonstrated as fit for purpose to identify and quantify 25 naturally occurring coumarin derivatives to meet the current product safety requirements. Satisfactory method accuracy and precision in complex essential oil sample matrices were demonstrated in the inter-laboratory validation study. The presented method was successfully implemented in our QC laboratories for high-throughput and cost-effective essential oil testing. Declarations Ackmowledgements We kindly thank a few colleagues from dōTERRA who contributed to this work, especially Nirmole Tambhar and Cecile Bascoul for the research of coumarins phototoxins, and Bryan Mauerman for providing the test samples. We also acknowledge Michael Scott, Sheila Stuligross, Megan Laryea-Akrong, Graham Curtin and Aaron Sorensen for their assistance. Compliance with ethical standards Ethical Approval This study does not involve human participants or animals. Conflict of Interest Siheng Li declares he has no competing interests. Colleen Kelly declares she has no competing interests. Radim Knob declares he has no competing interests. Kent McConnell declares he has no competing interests. Justin Barrett declares he has no competing interests. Funding No funding was received for this study. Author Contribution S.L.: planning and performing the experiments, writing - original draft. C.K.: performing the experiments, and writing – review and editing. R.K.: writing – review and editing. K.M.: performing the experiments. J.B.: Conceptualization, resources, writing – review and editing. Data Availability The data available in this study are available in Table 1; Figures 1, 2, 3, 4, 5, 6; and supplementary information. The datasets generated during this study are available from the corresponding author upon reasonable request. References Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ (2003) Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. J AOAC Int 86:412–431 Arigo A, Dugo P, Rigano F, Mondello L (2021) Linear retention index approach applied to liquid chromatography coupled to triple quadrupole mass spectrometry to determine oxygen heterocyclic compounds at trace level in finished cosmetics. J Chromatogr A 1649:462183 Cui D, Zhang Y, Duan X, Zhou Y, Li G, Feng X (2021) Progress in Pretreatment and Analytical Methods of Coumarins: An Update since 2012 – A Review. Crit Rev Anal Chem 51:503–526 Dugrand A, Olry A, Duval T, Hehn A, Froelicher Y, Bourgaud F (2013) Coumarin and furanocoumarin quantitation in citrus peel via ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS). J Agric Food Chem 61:10677–10684 European Commission (2009) Regulation (EC) no 1223/2009 of the european parliament and of the council of 30 November 2009 on cosmetic products. Off J Eur Union L 342:59–82 Fortune Business Insights (2021) Esseential oils market size, share & COVID-19 impact analysis, by type (citrus, eucalyptus, lavender, rosemary, tea tree, and others), by application (food & beverages, personal care & cosmetics, pharmaceuticals, and others), and regional forecast, 2021–2028. https://www.fortunebusinessinsights.com/industry-reports/essential-oils-market-101063 . Accessed 30 Jun 2022 Frérot E, Decorzant E (2004) Quantification of total furocoumarins in citrus oils by HPLC coupled with UV, fluorescence, and mass detection. J Agric Food Chem 52:6879–6886 Govindarajan R, Singh DP, Singh AP, Pandey MM, Rawat AKS (2007) A validated HPLC method for quantification and optimization of furocoumarins in different extracts of fruits of heracleum candicans. Chromatographia 66:401–405 IFRA (2008) Information Letter 799. 1–2 IFRA (2015) IFRA Standard 48th Amendement. Citrus oils and other furocoumarins containing essential oils: 1–2 International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (2005) ICH Harmonised Tripartite Guideline. Validation of analytical procedures: text and methodology Q2 (R1): 1–13 Khursheed A, Jain V (2021) Medicinal research progress of natural coumarin and its derivatives. J Nat Prod 11:648–662 Kreidl M, Rainer M, Jakschitz T, Bonn GK (2020) Determination of phototoxic furanocoumarins in natural cosmetics using SPE with LC-MS. Anal Chim Acta 1101:211–221 Kvittingen L, Sjursnes BJ, Schmid R (2021) Limonene in ctrus: a string of unchecked literature citings? J Chem Educ 98:3600–3607 Lin Y-K, Sheu M-T, Huang C-H, Ho H-O (2009) Development of a reversed-phase high-performance liquid chromatographic method for analyzing furanocoumarin components in citrus fruit juices and chinese herbal medicines. J Chromatogr Sci 47:211–215 Macmaster AP, Owen N, Brussaux S, Brevard H, Hiserodt R, Leijs H, Bast N, Weber B, Loesing G, Sherlock A, Schippa C, Vey M, Frérot E, Tissot E, Chaintreau A (2012) Quantification of selected furocoumarins by high-performance liquid chromatography and UV-detection: Capabilities and limits. J Chromatogr A 1257:34–40 Majors RE, Anastassiades M, Lehotay SJ (2010) The QuEChERS Revolution. LC GC Eur 23:418–429 Melough MM, Cho E, Chun OK (2018) Furocoumarins: a review of biochemical activities, dietary sources and intake, and potential health risks. Food Chem Toxicol 113:99–107 Melough MM, Chun OK (2018) Dietary furocoumarins and skin cancer: A review of current biological evidence. Food Chem Toxicol 122:163–171 Melough MM, Lee SG, Cho E, Kim K, Provatas AA, Perkins C, Park MK, Qureshi A, Chun OK (2017) Identification and quantitation of furocoumarins in popularly consumed foods in the U.S. using QuEChERS extraction coupled with UPLC-MS/MS analysis. J Agric Food Chem 65:5049–5055 Pereira TM, Franco DP, Vitorio F, Kummerle AE (2018) Coumarin compounds in medicinal chemistry: some important examples from the last years. Curr Top Med Chem 18:124–148 Prosen H, Kocar D (2008) Different sample preparation methods combined with LC-MS/MS and LC-UV for determination of some furocoumarin compounds in products containing citruses. Flavour Fragr J 23:263–271 Russo M, Rigano F, Arigò A, Dugo P, Mondello L (2021) Coumarins, psoralens and polymethoxyflavones in cold-pressed citrus essential oils: a review. J Essent Oil Res 33:221–239 Santana L, Uriarte E, Roleira F, Milhazes N, Borges F (2004) Furocoumarins in medicinal chemistry. synthesis, natural occurrence and biological activity. Curr Med Chem 11:3239–3261 Tisserand R, Young R (2014a) 2 - Essential oil composition. In: Tisserand R & Young R (eds) Essential Oil Safety (Second Edition). Churchill Livingstone, p 5–22 Tisserand R, Young R (2014b) 5 - The skin. In: Tisserand R & Young R (eds) Essential Oil Safety (Second Edition). Churchill Livingstone, p 69–98 Yang M, Fazio S, Munch D, Drumm P (2005) Impact of methanol and acetonitrile on separations based on π–π interactions with a reversed-phase phenyl column. J Chromatogr A 1097:124–129 Yuan L, Zhang D, Jemal M, Aubry A-F (2012) Systematic evaluation of the root cause of non-linearity in liquid chromatography/tandem mass spectrometry bioanalytical assays and strategy to predict and extend the linear standard curve range. Rapid Commun Mass Spectrom 26:1465–1474 Additional Declarations No competing interests reported. Supplementary Files FurocoumarinSupportingInfo063022.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3318708","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":230916219,"identity":"bd525083-5829-4ee7-802f-f91a307e663a","order_by":0,"name":"Siheng Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYDACdsaGAx/YSNLCzNh4cAYbgwQpWhiYD/OQpEW+mbnhsE2ZTR2/9NmHHxhqbKIJajE4zNhwOOdcmoRkX7qxBMOxtNwGglqYgVpy2w5LGJwBug7EJqhFvhmozBKoxf4MG/MPorQwgBzGCLKFh42NOFtAfjnYcy5NcsYZNjaLBGL8It/e/vjDjzIbfv4eNuYbH2psiHAYCkggTfkoGAWjYBSMAlwAAPJBOV7JZGovAAAAAElFTkSuQmCC","orcid":"","institution":"dōTERRA International","correspondingAuthor":true,"prefix":"","firstName":"Siheng","middleName":"","lastName":"Li","suffix":""},{"id":230916220,"identity":"0be26b34-dd90-430d-b4b2-7e9d0a1a23b3","order_by":1,"name":"Colleen Kelly","email":"","orcid":"","institution":"dōTERRA Global","correspondingAuthor":false,"prefix":"","firstName":"Colleen","middleName":"","lastName":"Kelly","suffix":""},{"id":230916221,"identity":"9ac79510-fc30-459f-b7cd-1b18745acd96","order_by":2,"name":"Radim Knob","email":"","orcid":"","institution":"dōTERRA International","correspondingAuthor":false,"prefix":"","firstName":"Radim","middleName":"","lastName":"Knob","suffix":""},{"id":230916222,"identity":"a6cc91ba-f242-43ab-8b28-10de6fb056e6","order_by":3,"name":"Kent McConnell","email":"","orcid":"","institution":"dōTERRA International","correspondingAuthor":false,"prefix":"","firstName":"Kent","middleName":"","lastName":"McConnell","suffix":""},{"id":230916223,"identity":"172d3e93-adfe-4006-987d-1e4ba2484a9d","order_by":4,"name":"Justin Barrett","email":"","orcid":"","institution":"dōTERRA International","correspondingAuthor":false,"prefix":"","firstName":"Justin","middleName":"","lastName":"Barrett","suffix":""}],"badges":[],"createdAt":"2023-09-02 03:44:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3318708/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3318708/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42847214,"identity":"9218f38b-5a17-457a-b6d9-989467a5636c","added_by":"auto","created_at":"2023-09-08 18:15:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":352730,"visible":true,"origin":"","legend":"\u003cp\u003eThe overlaid extracted-ion chromatogram and retention factor (k) of all target analytes on polar C18 (A), phenyl-hexyl (B), fluoro-phenyl (C) and biphenyl (D) column.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3318708/v1/604c75d1ca798a58be1a9972.png"},{"id":42847213,"identity":"17b1a5e1-6dd8-4c9b-b982-e2a84cf74e9d","added_by":"auto","created_at":"2023-09-08 18:15:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":308193,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The overlaid extracted-ion chromatograms of all target analytes in mobile phase pH at 3.5 (purple), 3.2 (blue), 3.0 (green) and 2.7 (orange). (B) The relative peak intensity of each target analytes in mobile phase pH at 3.5 (purple), 3.2 (blue), 3.0 (green) and 2.7 (orange).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3318708/v1/c0fef93941d1f6d82bd6e2ca.png"},{"id":42848333,"identity":"b781e9dd-ddac-4563-bfa0-e5d39413d167","added_by":"auto","created_at":"2023-09-08 18:23:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":197495,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative extracted ion chromatograms (XICs) of the molecular ion of 8-MOP, sphondin, bergapten and isobergapten (m/z 217.0); byakangelicin (m/z 335.1); phellopterin (m/z 301.0); epoxybergapmottin (m/z 355.2) (top); Representative XICs of the signature fragment ions of 8-MOP, sphondin, bergapten and isobergapten (m/z 202.0); byakangelicin (m/z 317.1); phellopterin (m/z 218.0); epoxybergapmottin (m/z 337.1) (bottom)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3318708/v1/926e248b7e316ef6948fa738.png"},{"id":42847219,"identity":"bd6dca2e-47a4-4528-827d-c2911bef02bb","added_by":"auto","created_at":"2023-09-08 18:15:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":180267,"visible":true,"origin":"","legend":"\u003cp\u003eLC elution profile of trioxsalen (I, SIM m/z 229.0), imperatorin and isoimperatorin (II, SIM m/z 271.0), and limonene (III, UV-Vis absorbance 206 nm) in furocoumarin fortified wild orange oil (blue trace) overlaid with furocoumarin solvent standard (green trace). The furocoumarin fortified wild orange oil was prepared by methanol extraction (A), 10% water in methanol extraction (B), and 10% water in methanol extraction followed by liquid-liquid extraction with hexane (C).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3318708/v1/9ef309af33f8e0e6a884068e.png"},{"id":42847216,"identity":"93e5374f-6cdf-4d49-8711-88d24994d7fd","added_by":"auto","created_at":"2023-09-08 18:15:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":258676,"visible":true,"origin":"","legend":"\u003cp\u003eMean recoveries of the 25 target analytes fortified in 19 types of essential oils at 5 µg/g. ND – The spiked recovery was not determined because of the high endogenous level of target analytes.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3318708/v1/85db77f01440edca63ecd0bf.jpg"},{"id":42848334,"identity":"983d07f5-5fcb-4f1b-b8c0-1024fcf62c94","added_by":"auto","created_at":"2023-09-08 18:23:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":118296,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Plot of mean recoveries and RSD of the 25 target analytes fortified in yuzu oil at four different concentration (5, 20, 40, 48 µg/g). The numerical results are presented in Table S2.\u0026nbsp; (B) Within-laboratory repeatability (RSD\u003csub\u003er\u003c/sub\u003e) versus between-laboratory reproducibility (RSD\u003csub\u003eR\u003c/sub\u003e) of the 25 target analytes in the yuzu oil fortified at 40 µg/g.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3318708/v1/22b7388794b096202f7e43d8.png"},{"id":43053667,"identity":"c6271cb3-246c-451a-bac9-38a1f632f2cc","added_by":"auto","created_at":"2023-09-13 09:07:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1719092,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3318708/v1/85ff6da3-894b-4d21-9219-2ea967f1a4c8.pdf"},{"id":42848335,"identity":"f5dbdf82-443b-4e1c-b678-25dde59d49a1","added_by":"auto","created_at":"2023-09-08 18:23:05","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":263978,"visible":true,"origin":"","legend":"","description":"","filename":"FurocoumarinSupportingInfo063022.docx","url":"https://assets-eu.researchsquare.com/files/rs-3318708/v1/aefb456b3d1230c536ec5389.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Method Development and Validation for the Analysis of Coumarin-based Phototoxins in Citrus-derived Essential Oils Using Liquid Chromatography-Mass Spectrometry","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoumarins belong to a family of naturally-occurring benzenoid lactone phytochemicals widely present in many plant species.(Pereira et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) They are secondary metabolites that typically are more concentrated in fruits and leaves, where they serve the purpose to expel insects and defend against fungal and bacterial infections. The variation of the functional groups during the plant biosynthesis results in the presence of numerous distinct coumarin-derivatives. The prominent sub-class of coumarin-derivatives is furocoumarins (also called furanocoumarins), which is defined by the core tricyclic structure of a furan ring unit fused onto a coumarin motif, such as angelicin, bergamottin, bergapten and psoralen.(Santana et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e)\u003c/p\u003e \u003cp\u003ePlant materials containing coumarin derivatives have been used in traditional health practices since ancient Egyptian times for a variety of benefits.(Khursheed and Jain \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) However, the photoallergenicity and phototoxicity of certain coumarin derivatives were gradually recognized with the advance of toxicological research. Human exposure to certain coumarin derivatives in the presence of long wavelength ultraviolet light (320 to 380 nm) can result in an acute skin allergy that may persist for several months.(Tisserand and Young \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e) Furthermore, furocoumarins are also reported as potential photo-mutagen and photo-carcinogen.(Melough et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) Due to adverse health effects, the level of several coumarin derivatives in natural herbal products are strictly regulated in many countries. Currently, the European regulation (EC) No 1223/2009 prohibits the use of furocoumarins except for the normal content in natural essences.(European Commission \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) In 2008, The International Fragrance Association (IFRA) added six furocoumarins (bergapten, bergamottin, byakangelicol, epoxybergamottin, isopimpinellin and oxypeucedanin) as regulated furocoumarin marker compounds, with their levels in finished cosmetics to no more than 5 ppm for leave-on products and 50 ppm for rinse off products.(IFRA \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) In 2015, the updated IFRA standard set the limit of bergapten in consumer essential oil products as no more than 15 ppm.(IFRA \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) The level of 6-methylcoumarin and 7-methoxycoumarin in fragrance products and cosmetics was also restricted by IFRA and the other major regulatory agencies.(Cui et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWith strong customer demand, the global essential oils market is projected to grow by 9.57% compound annual growth rate (CAGR) to 18.25\u0026nbsp;billion USD in 2028.(Fortune Business Insights \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) The total amount of essential oils produced worldwide has already reach 300,000 metric tons, with more than 70% of the essential oils used in fragrance, flavor and aromatherapy sector. Citrus-derived essential oils constituted 40% market share in the global essential oils market due to their availability and ideal organoleptic property. However, citrus plants are known to contain many coumarin derivatives.(Russo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) Citrus-derived essential oils are typically produced by cold pressing or steam distillation of the corresponding citrus plant materials. Thus, elevated levels of coumarin derivatives is not unusual in citrus oils, especially for essential oils produced by the cold-pressing of citrus peel, due to the facilitated co-extraction of the relatively polar and nonvolatile coumarins.(Tisserand and Young \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e) As secondary plant metabolites, the chemical profile of coumarin derivatives in herbal extracts is highly variable, which is dictated by genetic diversity and also by environmental factors such as growing season and geographical origin of the plants.(Fr\u0026eacute;rot and Decorzant \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) Considering the consumer essential oil products are commonly applied to the skin, it is critical to have a suitable analytical method for coumarin-based phototoxins in place as part of the quality control process to guarantee the essential oils are safe for consumers and meet regulatory safety requirements. Several furocoumarin analytical methods using various analytical techniques have been reported in the literature. They focus on a small subset of coumarin-derivatives in relatively simple sample matrices such as fruit and fruit juices.(Melough et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Govindarajan et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kreidl et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Prosen and Kocar \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) Due to the need for high-throughput quality control (QC) testing of essential oils, it is desirable to have a more comprehensive analytical method capable of monitoring all coumarin derivatives with safety concerns. The sample matrix of essential oils is highly complex and composed of concentrated phytochemicals. Based on our initial investigation, many reported analytical methods lack satisfactory method performance for these sample matrices. A 15-analyte high performance liquid chromatography (HPLC) method adopted by IFRA was intended for furocoumarins analysis in essential oils, but a long HPLC run time and limited capability to resolve matrix interferences in complex essential oils by UV detection hinder its wide application.(Macmaster et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Fr\u0026eacute;rot and Decorzant \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) Recently, other multi-analyte analytical methods have been reported but they have yet to be demonstrated with acceptable method reproducibility across laboratories.(Dugrand et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Arigo et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eHerein, we report the development of a multiplexed liquid chromatography-mass spectrometry (LC-MS) method to analyze 25 coumarin derivatives with known or potential safety concerns.(Melough and Chun \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Melough et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) The method development was mainly focused on the optimization of instrumental conditions and sample preparation to facilitate reliable analysis in the challenging matrix of essential oils. An inter-laboratory validation study was conducted in two laboratories in accordance with International Council of Harmonisation (ICH) guidelines on validation of analytical procedures and the methodology to demonstrate acceptable method performance in the essential oil matrix.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and materials\u003c/h2\u003e \u003cp\u003eMethanol (LCMS grade), hexane (HPLC grade) and formic acid (LCMS grade) were obtained from Millipore Sigma. Discovery\u003csup\u003e\u0026reg;\u003c/sup\u003e DSC-18 SPE bulk packing was purchased from Sigma-Aldrich.\u003c/p\u003e \u003cp\u003eA 1 mg/mL standard solution containing 15 furocoumarins (bergamottin, bergapten, byakangelicin, byakangelicol, epoxybergamottin, heraclenin, imperatorin, isoimperatorin, isopimpinellin, oxypeucedanin, oxypeucedanin hydrate, phellopterin, psoralen, 8-geranyloxypsoralen, 8-MOP) in acetonitrile was supplied by Chromadex (Irvine, CA, USA). The reference materials of citropten, 6-methylcoumarin, 7-methoxycoumarin and trioxalen were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bergaptol (\u0026gt;\u0026thinsp;97%) was obtained from Toronto Research Chemicals (Toronto, ON, Canada). The reference materials of 5-geranoxy-7-methoxycoumarin, angelicin, isobergapten, pimpinellin and sphondin were obtained from TargetMOL (Wellesley Hills, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStandard Preparation\u003c/h2\u003e \u003cp\u003eThe stock standard solution of each individual analyte was prepared at 800\u0026ndash;2000 \u0026micro;g/mL in appropriate solvent (methanol or ethanol) based on their solubility. A composite solution containing all 25 analytes at 100 \u0026micro;g/mL was prepared by mixing appropriate volumes of the stock standard solutions. The composite solution was further diluted to yield an intermediate stock solution at 20 \u0026micro;g/mL. The stock solution and intermediate stock solution were stored frozen for up to 3 months. Calibration standards containing each analyte at 0.04, 0.08, 0.20, 0.40 and 1.00 \u0026micro;g/mL were prepared by diluting the intermediate stock solution in 70/30 methanol/water (v/v).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSample Preparation\u003c/h2\u003e \u003cp\u003eThe essential oil samples used in this study were obtained from in-house production. A 1 g sample was weighed into a 10 mL volumetric flask and extracted with 10 mL 0.01% formic acid in 90/10 methanol/water (v/v), with 5 minutes sonication. The sample extract was transferred into a polypropylene centrifuge tube and centrifuged at 3000 \u0026times; g for 5 minutes. 1 mL aliquot of the top layer was transferred into a microcentrifuge tube and extracted with 50 \u0026micro;L hexane. After thorough mixing, the sample tube was centrifuged at 12000 \u0026times; g at 4\u0026deg;C for 5 minutes and the top hexane layer was discarded. The sample extract was transferred into a new microcentrifuge tube containing 50 mg DSC-18 SPE sorbent. After incubation at room temperature for 10 minutes, the sample extract was filtered (0.2 \u0026micro;m PTFE filter, Phenomenex) and an 80 \u0026micro;L aliquot was mixed with 920 \u0026micro;L 70/30 methanol/water (v/v) in an autosampler vial for LC-MS analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS analysis\u003c/h2\u003e \u003cp\u003eLC-MS analyses were performed on an Agilent 1290 Infinity LC system coupled with an Agilent 6125 MSD single-quadrupole mass spectrometer equipped with electrospray ionization (ESI) source. Data acquisition and processing were controlled by Agilent OpenLab software. Chromatographic separation was performed using a Phenomenex Kinetex\u003csup\u003e\u0026reg;\u003c/sup\u003e biphenyl column (100 \u0026times; 2.1 mm, 1.7 \u0026micro;m, 100 A). Mobile phase A was 0.01% formic acid in water and mobile phase B was methanol (LCMS Grade). The gradient elution program was as follows: 0\u0026ndash;4.0 min 50\u0026ndash;60%B, 4.0\u0026ndash;11.0 min 60\u0026ndash;95%B, 11.0\u0026ndash;12.0 min 95%B, 12.0-12.1 min 95\u0026thinsp;\u0026minus;\u0026thinsp;50%B, 12.1\u0026ndash;14.0 min 50%B. LC flow rate was 0.4 mL/min. The column oven was set to 40\u0026deg;C and the autosampler was at 5\u0026deg;C. The injection volume was 2 \u0026micro;L. The MS acquisition was carried out in positive ESI mode to monitor the SIMs in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The following MS source settings were applied: 340\u0026deg;C gas temperature, 11 L/min gas flow, 32 psi nebulizer gas, 4000 V capillary voltage.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe targeted coumarin derivatives, their partition coefficient (LogP) and their specific MS parameters for the identification and quantitation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnalyte\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMw\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLogP\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSIM 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFragmenter (V)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSIM 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFragmenter (V)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6-Methylcoumarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e92-48-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e160.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e161.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e105.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e220\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7-Methoxycoumarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e531-59-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e176.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e177.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e121.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsoralen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e66-97-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e186.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e187.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e131.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAngelicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e523-50-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e186.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e187.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e131.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBergaptol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e486-60-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e202.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e203.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e147.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitropten\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e487-06-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e206.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e207.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e192.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethoxsalen (8-MOP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e298-81-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e216.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e202.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e174.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e230\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSphondin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e483-66-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e216.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e202.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e174.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e230\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBergapten\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e484-20-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e216.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e202.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e174.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e230\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsobergapten\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e482-48-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e216.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e202.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e174.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e230\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrioxsalen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e3902-71-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e228.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e229.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e115.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e260\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsopimpinellin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e482-27-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e246.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e247.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e232.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePimpinellin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e131-12-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e246.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e247.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e232.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImperatorin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e482-44-0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e270.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e271.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e203.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsoimperatorin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e482-45-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e270.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e271.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e203.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxyimperatorin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e35740-18-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e286.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e287.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e203.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxypeucedanin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e3173-02-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e286.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e287.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e203.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhellopterin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e2543-94-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e300.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e218.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e301.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxypeucednin hydrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e2643-85-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e304.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e305.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e147.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e260\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eByakangelicol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e26091-79-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e316.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e231.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e317.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e130\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5-Geranoxy-7-methoxycoumarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e7380-39-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e328.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e193.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e329.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eByakangelicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e482-25-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e334.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e317.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e231.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8-Geranyloxypsoralen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e7437-55-0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e338.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e203.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e339.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBergamottin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e7380-40-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e338.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e203.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e339.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpoxybergamottin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e206978-14-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e354.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e337.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e355.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003ea\u003c/sup\u003e The partition coefficients (LogP) were obtained from Chemicalize database\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMethod Validation\u003c/h2\u003e \u003cp\u003eThe optimized analytical method was validated in accordance with ICH guidelines, including the evaluation of specificity, linearity, limit of quantitation (LOQ), accuracy, and precision.(International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) Yuzu oil was selected as a representative citrus-derived oil matrix because it was determined to be an analyte-free matrix. The linearity was determined by the correlation coefficients of the calibration curves. Specificity and LOQ were evaluated by the fortification of yuzu oil with each analyte at the lowest level to achieve the acceptable method performance. Accuracy and repeatability were evaluated by spiking yuzu oil with each analyte at four concentration levels (5, 20, 40 and 48 \u0026micro;g/g) in 3 to 6 replicates. The quantitation was performed using solvent calibration standards. The validation experiment was conducted in two doTERRA laboratories to evaluate reproducibility.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of Chromatographic and MS Conditions\u003c/h2\u003e \u003cp\u003eAll 25 target analytes presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e were selected based on current global regulatory requirements and potential safety concerns. In consideration of the low targeted analytical range, the complexity of essential oil sample matrix, and the necessity to differentiate each coumarin derivative, LC-MS was considered as the most appropriate analytical technique. Although LC-tandem mass spectrometry (LC-MS/MS) and LC-high resolution mass spectrometry (LC-HRMS) offer the most ideal selectivity and sensitivity, a properly developed LC-MS method in single ion monitoring (SIM) mode would provide sufficient method performance for this analysis. Furthermore, it is relatively easy to operate and maintain a single-quadrupole mass spectrometer and harmonize this method in multiple laboratories for quality control testing.\u003c/p\u003e \u003cp\u003eSuitable chromatographic conditions are essential to achieve optimal method specificity and sensitivity by minimizing matrix interference and resolving critical pairs of target analytes. In general, reversed-phase chromatography is amenable to the reasonable retention of coumarin derivatives. However, it is a challenge to accommodate 25 targeted coumarin derivatives with acceptable selectivity in a short LC analysis. In particular, the target analytes included six groups of isobaric analytes that are not feasible to be resolved by the MS detector, hence their selective identification fully relied on the resolving power of LC separation. To determine the most ideal analytical column for this method, the separation of the target analytes using UHPLC columns of different stationary phases was investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the chromatograms were obtained from the analyses on polar C18, phenyl-hexyl, fluoro-phenyl and biphenyl columns of the same dimensions (100 \u0026times; 2.1 mm) using identical LC gradients of water and methanol. The biphenyl column clearly offered the best retention for all the target analytes, attributed to the strong pi-pi interaction between the coumarin motif and the biphenyl stationary phase. Fewer analytes coeluted using the biphenyl column, with only three groups of coeluting clusters (6-methylcoumarin and psoralen; oxyimperatorin, isobergapten and pimpinellin; isoimperatorin and phellopterin) which can be resolved by MS detector based on their m/z difference of molecular ions. In comparison, the coelution of 8 to 14 analytes were observed using the other three types of columns. With a slight adjustment to a shallower LC gradient, baseline separation of all the critical analyte pairs was achieved, including the group of four isobaric analytes 8-MOP, sphondin, bergapten and isobergapten. Therefore, the biphenyl column was chosen to perform all the subsequent optimizations. The total analysis time of one injection was 14 minutes, which significantly improves testing throughput compared to many published methods.(Govindarajan et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Macmaster et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dugrand et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) However, it was noted that bergaptol was unable to be resolved from its isobaric isomer xanthotoxol on the biphenyl column. Bergaptol and xanthotoxol can either be quantified as a sum or will require an alternative LC separation approach.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA water-methanol mixture was used as eluent due to the incompatibility of acetonitrile with biphenyl stationary phase.(Yang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) To improve peak shape and facilitate the ionization, modifications to the mobile phase pH were investigated. The peak shape and retention time remained constant with the increase of mobile phase acidity, attributed to the lack of ionizable functional groups in the targeted coumarin-derivatives. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) However, it is noteworthy that the increased acid concentration actually suppressed the ionization process, resulting in lower MS response of certain analytes. In particular, the peak area of 7-methoxycoumarin was 20% higher in pH 3.5 compared to pH 2.7. Taking this into consideration, the addition of 0.01% formic acid as the additive of mobile phase A was sufficient for improved peak shape and optimal MS response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll of the target coumarin derivatives yielded stable protonated molecular ions [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e in positive electrospray ionization (ESI). Most of the molecular ions are well suited in the selected ion monitoring (SIM) to represent each target analyte. However, several molecular ions were less specific due to the high MS background and the susceptibility to matrix interference, including bergapten, sphondin, 8-MOP and isobergapten (m/z 217.0); phellopterin (m/z 301.0); byakangelicin (m/z 335.1); and epoxybergapmottin (m/z 355.2). (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) In order to find the most suitable ions in SIM acquisition to represent each of the target analytes, the fragmentor voltage was ramped up in 10 V increments from 70 V to 250 V to encourage collision-induced dissociation of the molecular ions. Through this process, we were able to identify the most specific signature fragment ions of each target analyte and their respective optimal fragmentor voltage. In particular, fragment ions of bergapten, sphondin, 8-MOP and isobergapten (m/z 202.0); phellopterin (m/z 218.0); byakangelicin (m/z 317.1); and epoxybergapmottin (m/z 337.1) were identified to offer significantly improved signal-to-noise ratio (S/N) compared to their molecular ions. The top two ions of each target analyte were selected as quantifier ion and qualifier ion in the SIM acquisition, which further improved the method specificity. The complete list of the SIM ions and fragmentor voltages are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The optimized chromatography and MS conditions enabled sensitive detection of most target analytes down to 10 to 20 pg on column. 7-methoxycoumarin and epoxybergamottin were the least sensitive analytes due to the higher background of their respective SIM ions. Overall, 40 pg on column was an achievable limit of detection (LOD) for all the target analytes with corresponding S/N more than 3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of Sample Preparation Procedure\u003c/h2\u003e \u003cp\u003eThe major constituents of citrus-derived essential oils are various monoterpenes, such as limonene, α-pinene, γ-terpinene and myrcene, with limonene as the most abundant constituent commonly above 50% (w/w).(Kvittingen et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) Although monoterpenes are hydrocarbons that are not ionizable by ESI, they share elution profiles similar to coumarin derivatives in reversed-phase chromatography. To understand whether the analysis of coumarin-derivatives is affected by the presence of concentrated limonene in the citrus-derived oil, distilled wild orange oil fortified with the target analytes was dissolved in methanol and analyzed by a LC-UV-MS system, with UV detection at the apex of limonene UV-Vis spectrum (206 nm). Although most of the target analytes were not affected, suppressed response and retention time shifts were observed in the elution of imperatorin (8.5 min), trioxsalen (8.6 min) and isoimperatorin (9.3 min), which happened to overlap with the elution region of limonene (8.3\u0026ndash;8.8 min) (Fig.\u0026nbsp;4AI-AIII). Column performance and MS ionization were compromised by the overloading of an abundance of limonene in the tested sample. Thus, additional sample clean-up to remove limonene is required for the accurate analysis of all the targeted coumarin-derivatives.\u003c/p\u003e \u003cp\u003eQuEChERS is the most widely used sample preparation in food and environmental applications due to excellent cost-effectiveness.(Anastassiades et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Majors et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) It is the primary solution to extract organic residues from many food matrices, such as protein, carbohydrates and lipids. However, it is ineffective at selectively extracting target analytes from small molecule matrix components with similar partition coefficients. The calculated partition coefficient of limonene (logP\u0026thinsp;=\u0026thinsp;3.22) is within the range of the partition coefficients of the target analytes (logP 1.0\u0026ndash;4.8), indicating a tendency of QuEChERS to co-extract both the analytes and limonene into the acetonitrile phase (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thus, an alternative technique to remove the monoterpene matrix was explored for this analysis.\u003c/p\u003e \u003cp\u003eThe use of an appropriate, selective extraction solvent turned out to be a more suitable sample preparation technique. Although limonene is soluble in medium polarity solvents such as methanol and acetonitrile, the use of water as a co-solvent effectively minimizes its solubility. On the other hand, coumarin derivatives are relatively more soluble in a methanol-water mixture. A water-methanol mixture as the extraction solvent is capable of extracting coumarin derivatives in the presence of monoterpene sample matrix. 10% water in methanol (v/v) was determined as the optimal ratio in removing abundant limonene while maintaining acceptable recovery of all target analytes. After centrifugation, most of the sample matrix precipitated while the target analytes extracted into the upper layer of the extraction solvent. Further increases of water ratio marginally improved the removal of limonene but resulted in much higher recovery loss of the least polar analytes, such as bergamottin and 5-geranoxy-7-methoxycoumarin.\u003c/p\u003e \u003cp\u003eFor a sample containing\u0026thinsp;\u0026gt;\u0026thinsp;90% limonene, 10% water in methanol (v/v) as the extraction solvent is not sufficient to remove all the matrix interference for LC-MS analysis (Fig.\u0026nbsp;4BI-III). To further improve limonene removal, an additional step of liquid-liquid extraction with hexane was introduced. A sample diluted with 10% methanol was mixed with hexane in a 1:20 ratio, then it was partitioned by centrifugation and the hexane layer was discarded. This enabled the removal of the matrix interference associated with residual limonene in the sample extract. As shown in Fig.\u0026nbsp;4III, the limonene level in the wild orange oil sample extract after liquid-liquid extraction was significantly reduced compared to the methanol/water extract. The retention time of trioxsalen, imperatorin and isoimperatorin in the wild orange oil sample extract also overlapped well with a solvent standard of the same concentration, indicating the successful removal of limonene interference (Fig.\u0026nbsp;4CI-II). Subsequently, the sample was incubated with C18-based dispersive solid phase extraction (dSPE) sorbent to remove residual hexane that can cause retention time shifts.\u003c/p\u003e \u003cp\u003eThe optimized extraction procedure was evaluated through the spiked recovery of target analytes in various essential oils at 5 \u0026micro;g/g, which is the intended LOQ of this analysis. Nineteen essential oils were screened as representative of herbal extracts derived from various citrus peels, leaves and flowers, as well as from the similar botanical family of Rutaceae and Apiaceae with reported phototoxicity. In general, the spiked recoveries were mostly within the acceptable range (75\u0026ndash;120%), as represented by the green color in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Several essential oils produced from citrus peels contain measurable levels of targeted coumarin-derivatives that hinder spiked recovery determination at 5 \u0026micro;g/g fortification level. Thus, their recoveries were not determined (ND). Out of these 25 targeted compounds, epoxybergamottin, bergamottin, 8-geranyloxypsoralen and 5-geranoxy-7-methoxycoumarin showed lower spiked recovery. The high lipophilicity of epoxybergamottin (LogP\u0026thinsp;=\u0026thinsp;3.8), bergamottin (LogP\u0026thinsp;=\u0026thinsp;4.8), 8-geranyloxypsoralen (logP\u0026thinsp;=\u0026thinsp;4.8) and 5-geranoxy-7-methoxycoumarin (LogP\u0026thinsp;=\u0026thinsp;4.5) was presumably causing their recovery loss during the sample extraction. Overall, this optimized sample extraction procedure was demonstrated to be applicable for various types of essential oils.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMethod Validation\u003c/h2\u003e \u003cp\u003eAn inter-laboratory method validation study was conducted in accordance with ICH guidelines on validation of analytical procedures.(International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) Based on ICH guidelines, this method is considered as a category II Quantitative Impurity Assay, thus the method performance characteristics evaluated as part of the validation study included specificity, linearity, range, limit of quantitation (LOQ), accuracy and precision.\u003c/p\u003e \u003cp\u003eThe method specificity was investigated by comparing a blank sample with the same blank sample spiked with the target analytes. The selection of a blank citrus essential oil free of coumarin derivatives was rather limited because of naturally occurring coumarin derivatives inherent in most of citrus oils. Through our initial sample screening, yuzu oil was chosen as a representative blank sample without detectable amounts of target analytes. All the primary SIMs were shown to be highly selective with no chromatographic interference observed in yuzu oil. The chromatograms are presented in supporting information (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The secondary SIMs were less selective and used as qualifier ions for confirmation purpose. Overall, successful analyte identification was demonstrated at concentrations equal to or above the method LOQ.\u003c/p\u003e \u003cp\u003eThe analytical range evaluated in this study was from 0.04 \u0026micro;g/mL to 1 \u0026micro;g/mL, which was equivalent to the range of 5 \u0026micro;g/g to 125 \u0026micro;g/g in the essential oil sample. Calibration curves were established by a set of five calibration standards using quadratic regression and 1/x weighting. The quadratic standard curves provided a better fit due to a non-linear response near the upper end of the calibration curves, presumably related to saturation during ionization.(Yuan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) Over the course of the validation study, the correlation coefficient (r) of all target analytes were consistently above 0.99, which demonstrates acceptable linearity of this method (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to the absence of citrus-derived oils with certified levels of coumarin derivatives, the method accuracy was evaluated by the marginal recovery% of the blank sample fortified with target analytes at four concentration levels (5 \u0026micro;g/g, 20 \u0026micro;g/g, 40 \u0026micro;g/g and 48 \u0026micro;g/g). Repeatability (RSD\u003csub\u003er\u003c/sub\u003e) was determined from the analysis of the fortified sample in three to six replicates, and the inter-laboratory reproducibility (RSD\u003csub\u003eR\u003c/sub\u003e) was determined from the repeatability results from two independent laboratories. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and Table S2, acceptable recoveries and RSDs were obtained in the validation study. The mean recoveries were within 80\u0026ndash;115%, except bergamottin and 5-geranoxy-7-methoxycoumarin. The lower mean recoveries in the range of 60\u0026ndash;80% were presumably due to recovery loss during the extraction because of their high lipophilicity. The RSD\u003csub\u003er\u003c/sub\u003e of all the analytes at 5 \u0026micro;g/g fortification level was between 4\u0026ndash;10%. In higher fortification levels, the analyses were more precise with RSD\u003csub\u003er\u003c/sub\u003e of all 25 targeted analytes below 5%. The precision results obtained from two laboratories was also aligned with the single-laboratory results, with RSD\u003csub\u003eR\u003c/sub\u003e below 6% for all the analytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The narrow distribution of RSD\u003csub\u003er\u003c/sub\u003e and RSD\u003csub\u003eR\u003c/sub\u003e demonstrates the high consistency and precision of this analytical method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe LOQs were determined as the lowest fortification level that met the identification criteria and showed acceptable recovery and precision. Yuzu oil fortified at 5 \u0026micro;g/g had a S/N consistently above 10 for all 25 analytes in the two laboratories, as shown in Table S3. The spike recovery and precision also met their respective acceptance criteria. Therefore, the LOQ of this analytical method is set as 5 \u0026micro;g/g.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA multiplexed LC-MS method was developed and thoroughly validated for the analysis of coumarin-derivatives in essential oils. This method enables the analysis of many important coumarin-based phototoxins in essential oils by using simple sample preparation and easy to operate instrumentation. An optimized extraction solvent was used to extract the coumarin-derivatives and remove the limonene matrix interference simultaneously. The method was demonstrated as fit for purpose to identify and quantify 25 naturally occurring coumarin derivatives to meet the current product safety requirements. Satisfactory method accuracy and precision in complex essential oil sample matrices were demonstrated in the inter-laboratory validation study. The presented method was successfully implemented in our QC laboratories for high-throughput and cost-effective essential oil testing.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAckmowledgements\u003c/h2\u003e\n\u003cp\u003eWe kindly thank a few colleagues from dōTERRA who contributed to this work, especially Nirmole Tambhar and Cecile Bascoul for the research of coumarins phototoxins, and Bryan Mauerman for providing the test samples. We also acknowledge Michael Scott, Sheila Stuligross, Megan Laryea-Akrong, Graham Curtin and Aaron Sorensen for their assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompliance with ethical standards\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthical Approval \u0026nbsp;\u003c/em\u003eThis study does not involve human participants or animals.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConflict of Interest\u003c/em\u003eSiheng Li declares he has no competing interests. Colleen Kelly declares she has no competing interests. Radim Knob declares he has no competing interests. Kent McConnell declares he has no competing interests. Justin Barrett declares he has no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contribution\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.L.: planning and performing the experiments, writing - original draft. C.K.: performing the experiments, and writing \u0026ndash; review and editing. R.K.: writing \u0026ndash; review and editing. K.M.: performing the experiments. J.B.: Conceptualization, resources, writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data available in this study are available in Table 1; Figures 1, 2, 3, 4, 5, 6; and supplementary information. The datasets generated during this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ (2003) Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. 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J Essent Oil Res 33:221\u0026ndash;239\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantana L, Uriarte E, Roleira F, Milhazes N, Borges F (2004) Furocoumarins in medicinal chemistry. synthesis, natural occurrence and biological activity. Curr Med Chem 11:3239\u0026ndash;3261\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTisserand R, Young R (2014a) 2 - Essential oil composition. In: Tisserand R \u0026amp; Young R (eds) Essential Oil Safety (Second Edition). Churchill Livingstone, p\u0026nbsp;5\u0026ndash;22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTisserand R, Young R (2014b) 5 - The skin. In: Tisserand R \u0026amp; Young R (eds) Essential Oil Safety (Second Edition). Churchill Livingstone, p\u0026nbsp;69\u0026ndash;98\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang M, Fazio S, Munch D, Drumm P (2005) Impact of methanol and acetonitrile on separations based on π\u0026ndash;π interactions with a reversed-phase phenyl column. J Chromatogr A 1097:124\u0026ndash;129\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan L, Zhang D, Jemal M, Aubry A-F (2012) Systematic evaluation of the root cause of non-linearity in liquid chromatography/tandem mass spectrometry bioanalytical assays and strategy to predict and extend the linear standard curve range. Rapid Commun Mass Spectrom 26:1465\u0026ndash;1474\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"coumarins, furocoumarins, essential oils, LC-MS","lastPublishedDoi":"10.21203/rs.3.rs-3318708/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3318708/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The occurrence of coumarin-based photoactive compounds in essential oil has been a concern due to the adverse health effects it has on human health. A fit-for-purpose analytical method for major coumarin-based phototoxins is critical to guarantee the essential oils product safety and compliance with the regulatory requirements. Herein, a liquid chromatography-mass spectrometry (LC-MS) analytical method was developed to identify and quantify 25 coumarin derivatives in essential oils. The sample preparation procedure was optimized for essential oils to use selective solvent extraction to remove the limonene matrix interference. To achieve the optimal sensitivity and specificity, UHPLC column of biphenyl stationary phase was used for this analysis. An inter-laboratory validation study was conducted to evaluate the method performance. The limit of quantification (LOQ) for all target analytes were validated at 5 µg/g. Satisfactory recoveries were obtained for the majority of analytes in the range of 80-115%. Both the repeatability RSDr (within-laboratory) and reproducibility RSDR (between-laboratory) \u003c10%. This method was demonstrated as applicable for the quality control testing of coumarin-based phototoxins in essential oils.","manuscriptTitle":"Method Development and Validation for the Analysis of Coumarin-based Phototoxins in Citrus-derived Essential Oils Using Liquid Chromatography-Mass Spectrometry","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-08 18:15:00","doi":"10.21203/rs.3.rs-3318708/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c11d073-7e76-45fe-adbb-b6b5c4bbe25f","owner":[],"postedDate":"September 8th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-20T09:29:13+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-08 18:15:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3318708","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3318708","identity":"rs-3318708","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}