Concurrent identification of 11 major primary active compounds in Huangqin Qingfei Decoction by liquid chromatography tandem mass spectrometry via liquid chromatography tandem mass spectrometry.

Huangqin Qinfei Decoction (HQD) is a traditional Chinese medicine that is administered for acute pneumonia, bronchial inflammation, acute bronchitis and acute lung infection. In this study, we used liquid chromatography linked with tandem mass spectrometry (LC-MS/MS) for the concurrent identification of 11 bioactive compounds; namely, baicalin, baicalein, wogonoside, scutellarin, wogonin, oroxylin A, geniposide, genipin, geniposidic acid, chlorogenic acid, and crocin-I, for the quality control of HQD. The evaluation was conducted on an Agilent Poroshell 120 EC-C18 (2.1mm×100mm, 2.7µm) with gradient elution in the mobile phase with 0.1% formic acid and 1mM/L ammonium acetate in water as solvent A and methanol as solvent B at a flow rate of 0.3mL/min in under 12 min. Mass spectrometric detection was conducted in the selected reaction monitoring mode utilizing electro spray ionization in the positive and negative modes. Every one of the calibration curves had good linearity with R2 >0.9992. Intra-day and inter-day accuracies for every one of the evaluated components were expressed as the relative standard deviation (RSD) from 1.72%-5.02% and 0.63%-5.99%, respectively. The recuperation of the 11 compounds that were measured at the three concentrations was within 94.05%-105.18%, with the RSD ≤ 6.26%. The use of this method was determined through the effective evaluation of 11 compounds in 5 batches of HQD. The confirmed method is precise, sensitive, and effective for identifying the contents of the chosen compounds in HQD for quality control.

An oligosaccharide-marker approach to quantify specific polysaccharides in herbal formula by LC-qTOF-MS: Danggui Buxue Tang, a case study.

Polysaccharides have broad bioactivities and are major components of water decoction of herb formulae. However, the quality control of polysaccharides remains a challenge. Oligosaccharide-fragment approach has been considered in elucidating chemical structures of polysaccharides, but never been used for quantitation. Using reference chemicals and a real sample Danggui Buxue Tang (DBT) in this study, an oligosaccharide-marker approach was established to quantify specific polysaccharides. Firstly, linear relationships between parent polysaccharides and hydrolysis-produced daughter oligosaccharides were verified using reference polysaccharides. Then in case of DBT, two fluorescence-labeled oligosaccharides with high specificity to individual parent polysaccharides were selected as markers. They were easily isolated and identified. Their potential in quantification of parent polysaccharides were satisfactorily validated in terms of linearity (r≥0.99), repeatability (RSD ≤ 8.4 %), and spike recovery (≥80 %). This method could be a promising approach for quality assessment of polysaccharides in herbal formulae.

Combining untargeted and targeted metabolomics approaches for the standardization of polyherbal formulations through UPLC-MS/MS.

INTRODUCTION: Polyherbal formulations are an integral part of various indigenous medicinal systems such as Traditional Chinese Medicine (TCM) and Ayurveda. The presence of a very large number of compounds makes the quality control of polyherbal formulations very difficult. OBJECTIVES: To overcome this problem, we have developed a comprehensive strategy for the dereplication of natural products in polyherbal formulations by using Adhatoda vasica as a case study. METHODS: The strategy is based on five major steps: the collection of plant samples from different locations to observe the effects of environmental variables; LC-ESI-MS/MS-based untargeted metabolite profiling of the plant samples to identify marker compounds using extensive chemometric analysis of the obtained data; the identification of marker compounds in polyherbal products; the isolation, purification and characterization of the marker compounds; and MRM-based quantitative analysis of the isolated marker compounds using LC-ESI-MS/MS. RESULTS: Using this strategy, we identified a total of 51 compounds in the methanolic extract of A. vasica plants from 14 accessions. Chemical fingerprinting of the plant led to the identification of characteristic peaks that were used to confirm the presence of A. vasica in complex polyherbal formulations. Four quinazoline alkaloids (marker compounds) were isolated, purified and quantified in various herbal formulations containing A. vasica. CONCLUSION: This method demonstrates a comprehensive strategy based on untargeted and targeted metabolite analysis that can be used for the standardization of complex polyherbal formulations.

Multiclass mycotoxins in lotus seeds analysed by an isotope-labelled internal standard-based UPLC-MS/MS.

OBJECTIVES: This study aimed to explore the residue levels of multiclass mycotoxins in medicinal and edible lotus seeds. METHODS: A rapid and reliable isotope-labelled internal standard-based UPLC-MS/MS method was developed and validated for sensitive and accurate analysis of multiclass mycotoxins including aflatoxins (AFB1 , AFB2 , AFG1 and AFG2 ), ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON), fumonisins (FB1 and FB2 ), T-2 and HT-2 toxins in lotus seeds. Some critical conditions such as extract solution with the addition of isotope-labelled internal standard, type of mobile phase and the elution condition were scientifically optimized. The 11 mycotoxins obtained satisfactory resolution and sensitive detection in multiple reactions monitoring scanning mode combined with the ion switching technology in positive and negative ion switching mode. KEY FINDINGS: The developed isotope-labelled internal standard-based UPLC-MS/MS method exhibited an approving linearity (r ≥ 0.9984), high sensitivity (limit of detection in the range of 0.015-30.05 µg/kg), acceptable precision (RSDs ≤6.3%) and good recovery (76.0-116.0%) for 11 analytes, respectively. Ten batches of real lotus seed samples were tested, and three batches out of which were contaminated with AFB1 , FB2 , T-2 and ZEN. AFB1 showed the highest occurrence rate (30%) with contents of 10.50 and 8.32 µg/kg in two samples over the official limit (5.0 µg/kg). CONCLUSIONS: The monitoring of multiclass mycotoxins in Chinese herbal medicines is in great urgency to ensure the security of consumers. The proposed method could be further utilized for simple, sensitive and rapid detection of more mycotoxins in other complex matrices to compensate for matrix effects.

Development and validation of a UFLC-MS/MS method for simultaneous quantification of sixty-six saponins and their six aglycones: Application to comparative analysis of red ginseng and white ginseng.

A new and sensitive ultra fast liquid chromatography coupled with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC-MS/MS) method was developed to evaluate the quality of Red ginseng (RG) and to find out its chemical markers by comparing with multi-batches of RG and white ginseng (WG). This innovative method could quantify sixty-six saponins and their six aglycones including 10 pairs of 20(S) and 20(R) epimers within 35 min simultaneously. All compounds could be determined in individual multiple-reaction monitoring channel without interference, and the optimized method was rapid, accurate, precise, reproducible and efficient. Using the orthogonal partial least squared discriminant analysis, ginsenosides Rg5, Rh4, Rk1, Rs4, F4, and 20(S)-Rg3 were found to be the characteristic components of RG, the six compounds should be suggested as quality control markers to distinguish RG from WG. These findings will be significant for standardizing the processing procedures of RG and ensuring the consistent quality, as well as consequently the efficacy of RG in clinical applications. Results will be helpful in providing crucial chemical profiles of RG.

Rapid methodology via mass spectrometry to quantify addition of soybean oil in extra virgin olive oil: A comparison with traditional methods adopted by food industry to identify fraud.

Fast and innovative methodology to monitors the addition of soybean oil in extra virgin olive oil was developed employing ESI-MS with ionization operating in positive mode. A certified extra virgin olive oil and refined soybean oil samples were analyzed by direct infusion, the identification of a natural lipid marker present only in soybean oil (m/z 886.68 [TAG+NH4]+) was possible. The certified extra virgin olive oil was purposely adulterated with soybean oil in different levels (1, 5, 10, 20, 50, 70, 90%) being possible to observe that the new methodology is able to detect even small fraud concentration, such as 1% (v/v). Additionally, commercial samples were analyzed and were observed the addition of soybean oil as a common fraud in this segment. This powerful analytical method proposed could be applied as routine analysis by control organization, as well as food industries, considering its pronounced advantages; simplicity, rapidity, elevated detectability and minor amounts of sample and solvent consumed.

LC-MS/MS analysis and evaluation of the anti-inflammatory activity of components from BushenHuoxue decoction.

CONTEXT: BushenHuoxue decoction (BSHXD) is a Chinese medicine prescription, which is composed of nine Chinese medical materials, used to treat osteoarthritis (OA). OBJECTIVE: This study develops sensitive and convenient LC-MS/MS methods to analyze chemical components from BSHXD, and assess the anti-inflammatory activities thereof. MATERIALS AND METHODS: The chemical composition from BSHXD water extract was qualitative analyzed by high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS). Twelve reference compounds were analyzed by UPLC-ESI-MS/MS. Anti-inflammatory activities of target components were assessed by ELISA at 20 and 100 µg/mL. RESULTS: It is the first time that 88 compounds were qualitatively identified from BSHXD, of which 12 with potential in treating OA according to the literature were quantified. Within BSHXD the contents of quercetin, isopsoralen, icarisideII, osthole, and isoimperatorin increased remarkably compared with those in single herb which make up BSHXD, the contents were 0.1999, 0.4634, 0.0928, 0.5364, and 0.1487 mg/g. ELISA data displayed that BSHXD and the five compounds mentioned inhibited the expressions of TNF-α, IL-6 and NO released from LPS-stimulated RAW264.7 cell, with maximum inhibition rates of 104.05% (osthole, 100 µg/mL), 100.03% (osthole, 100 µg/mL), and 93.46% (isopsoralen, 20 µg/mL), respectively. DISCUSSION AND CONCLUSION: Content changes of 12 compounds in BSHXD and single herbs which comprise the prescription were measured and analyzed. Contents of five compounds increased may be explained by solubilization between drugs and chemical reaction. ELISA results reported that the increased contents of the five compounds could inhibit expression of the inflammatory factors.

Validated quantification method for five ephedrines in dietary supplements using LC-MS/MS: Application to 503 cases.

Ephedrines in dietary supplements can arise from herbs or illegal adulteration so a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for separation and quantification of ephedrine, pseudoephedrine, norephedrine, norepseudoephedrine, and methyl-ephedrine, some of which are isomer pairs of pseudo-structures. This method includes liquid-liquid extraction of ephedrines from three typical dietary supplement matrices-solid, liquid, and oil-as well as liquid chromatographic separation. After liquid chromatographic separation, ephedrines are qualitatively and quantitatively analyzed using triple quadrupole mass spectrometry with positive electrospray ionization in multi-reaction monitoring (MRM) mode. Ephedrine recoveries in a solid matrix ranged from 53.3-91.5%, in a liquid matrix from 56.4-102.3%, and in an oil matrix from 51.7-01.2%. Linearity ranges were 10-1000ng/g in solid and oil matrix and 1-100ng/ml in liquid matrix. Accuracy was -11.5-16.3%. Intra-day and inter-day variation are less than 5.9% and 7.3%, respectively. Expanded uncertainty of quantification is less than 0.123ng/g in a solid matrix, less than 0.139ng/ml in a liquid matrix, and less than 0.158ng/g in an oil matrix. Data collected for more than 500 routine samples are presented and discussed.

Rapid Identification and Assignation of the Active Ingredients in Fufang Banbianlian Injection Using HPLC-DAD-ESI-IT-TOF-MS.

Fufang Banbianlian Injection (FBI) is a well-known traditional Chinese medicine formula composed of three herbal medicines. However, the systematic investigation on its chemical components has not been reported yet. In this study, a high-performance liquid chromatography combined with diode-array detector, and coupled to an electrospray ionization with ion-trap time-of-flight mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) method, was established for the identification of chemical profile in FBI. Sixty-six major constituents (14 phenolic acids, 14 iridoids, 20 flavonoids, 2 benzylideneacetone compounds, 3 phenylethanoid glycosides, 1 coumarin, 1 lignan, 3 nucleosides, 1 amino acids, 1 monosaccharides, 2 oligosaccharides, 3 alduronic acids and citric acid) were identified or tentatively characterized by comparing their retention times and MS spectra with those of standards or literature data. Finally, all constituents were further assigned in the individual herbs (InHs), although some of them were from multiple InHs. As a result, 11 compounds were from Lobelia chinensis Lour, 33 compounds were from Scutellaria barbata D. Don and 38 compounds were from Hedyotis diffusa Willd. In conclusion, the developed HPLC-DAD-ESI-IT-TOF-MS method is a rapid and efficient technique for analysis of FBI sample, and could be a valuable method for the further study on the quality control of the FBI.

Sensitive quantification of six steroidal lactones in Withania coagulans extract by UHPLC electrospray tandem mass spectrometry.

A method for the concurrent determination of six known steroidal lactones (syn. withanolides or withasteroids), namely withaferin A, withanolide H, withanolide K, withanolide A, withacoagulin H, and withanolide J in Withania coagulans extracts was developed. Extracts of Withania species and purified withanolides are considered among the most important natural products used for medicinal purposes. Methanolic extract of plant material was subjected to reverse phase ultra-high performance liquid chromatography (UHPLC) coupled with electrospray (JetStream ESI) triple quadrupole mass spectrometer operated in the Multiple Reaction Monitoring (MRM) mode. Satisfactory separation of withanolide component was achieved within 9 min on UHPLC runtime. The limits of detection (LOD) and the limits of quantitation (LOQs) for the six withanolides ranged between 0.040-4.80 ng/mL, and 0.13-16 ng/mL, respectively. Linear responses were attained for all six withanolides in two orders of magnitude with the linear regression coefficient values ⩾0.998. At the five QC levels inspected, the relative standard deviations (RSD) were found below 5% in most cases. The newly developed method is fast, precise, and sensitive, therefore, the method can be used for high-throughput quantification of various withanolides in W. coagulans extract, and other herbal formulations, derived from W. coagulans.

Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography-triple quadrupole mass spectrometry.

Buxus papillosa is one of the most extensively studied species of the genus Buxus known to possess steroidal alkaloids which can be used for assessing the various pharmacological activities of this plant. This paper describes the liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (LC-ESI-QQQ-MS) method for the quantification of six steroidal alkaloids as chemical markers in the extracts of leaves, roots and stems of B. papillosa. Quantitative MS/MS analysis was carried out by optimization of the most sensitive transition for each analyte. This has yielded detection and quantification limits of 0.486-8.08 ng/mL and 1.473-24.268 ng/mL, respectively for all analytes. Linearity of response was also achieved and the regression coefficient found to be >0.99 for all analyzed compounds. The newly developed MRM (Multiple Reaction Monitoring) method showed excellent sensitivity for the quantification of steroidal alkaloids within 15 min run time. This paper describes the application of LC-QQQ-MS technique for steroidal alkaloids analysis in plant samples.

Determination of short chain carboxylic acids in vegetable oils and fats using ion exclusion chromatography electrospray ionization mass spectrometry.

A new method for quantification of short chain C1-C6 carboxylic acids in vegetable oils and fats by employing Liquid Chromatography Mass Spectrometry (LC-MS) has been developed. The method requires minor sample preparation and applies non-conventional Electrospray Ionization (ESI) liquid phase chemistry. Samples are first dissolved in chloroform and then extracted using water that has been spiked with stable isotope labeled internal standards that are used for signal normalization and absolute quantification of selected acids. The analytes are separated using Ion Exclusion Chromatography (IEC) and detected with Electrospray Ionization Mass Spectrometry (ESI-MS) as deprotonated molecules. Prior to ionization the eluent that contains hydrochloric acid is modified post-column to ensure good ionization efficiency of the analytes. The averaged within run precision and between run precision were generally lower than 8%. The accuracy was between 85 and 115% for most of the analytes. The Lower Limit of Quantification (LLOQ) ranged from 0.006 to 7mg/kg. It is shown that this method offers good selectivity in cases where UV detection fails to produce reliable results.

Ultrahigh-performance liquid chromatography-ion trap mass spectrometry characterization of the steroidal saponins of Dioscorea panthaica Prain et Burkill and its application for accelerating the isolation and structural elucidation of steroidal saponins.

Dioscorea panthaica is a traditional Chinese medicinal herb used in the treatment of various physiological conditions, including cardiovascular disease, gastropathy and hypertension. Steroidal saponins (SS) are the main active ingredients of this herb and have effects on myocardial ischemia and cancer. The phytochemical evaluation of SS is both time-consuming and laborious, and the isolation and structural determination steps can be especially demanding. For this reason, the development of new methods to accelerate the processes involved in the identification, isolation and structural elucidation of SS is highly desirable. In this study, a new ultrahigh performance liquid chromatography-ion trap mass spectrometry (UHPLC-IT/MS(n)) method has been developed for the identification of the SS in D. panthaica Prain et Burkill. Notably, the current method can distinguish between spirostanol and furostanol-type compounds based on the fragmentation patterns observed by electrospray ionization-ion trap mass spectrometry (ESI-IT/MS(n)) analysis. UHPLC-IT/MS(n) was used to conduct a detailed investigation of the number, structural class and order of the sugar moieties in the sugar chains of the SS present in D. panthaica. The established fragmentation features were used to analyze the compounds found in the 65% ethanol fraction of the water extracts of D. panthaica. Twenty-three SS were identified, including 11 potential new compounds and six groups of isomers. Two of these newly identified SS were selected as representative examples, and their chemical structures were confirmed by (1)H and (13)C NMR analyses. This newly developed UHPLC-IT/MS(n) method therefore allowed for the efficient identification, isolation and structural determination of the SS in D. panthaica.

Relative bioavailability of gastrodin and parishin from extract and powder of Gastrodiae rhizoma in rat.

A rapid, sensitive and reliable UHPLC-ESI-MS/MS method was developed for simultaneous determination of gastrodin and parishin in rat plasma. The LLOQ of the two analytes were 1.00×10(-1) and 8.30×10(-5)µg/mL, respectively. The intra-day and inter-day precision were all less than 10% of the relative standard deviation (RSD), whilst the accuracy were all within ±15% of the relative error (RE). The proposed method was successfully applied for pharmacokinetics study on the two analytes in rats after oral administration of Gastrodiae rhizoma (GR) extract and powder at low, medium and high dosages. Blood samples were collected from the suborbital vein at predetermined time points and were precipitated using methanol. Chromatographic separations were carried out on a Kinetex XB-C18 column (2.1mm×150mm, 1.7µm) with a gradient mobile phase of acetonitrile-water with 0.1% formic acid as a modifier. The pharmacokinetic parameters of the two analytes in rats were obtained and the relative bioavailability of gastrodin and parishin in two formulations were calculated. The results indicated that higher bioavailability was obtained when low dosage of GR powder was used, whereas, higher bioavailability values were obtained when medium and high dosages of GR extract were used.

Comparative investigation of in vitro biotransformation of 14 components in Ginkgo biloba extract in normal, diabetes and diabetic nephropathy rat intestinal bacteria matrix.

Most herbal medicines will be metabolized by intestinal bacteria in the gastrointestinal tract before absorbed by the small intestine. Ginkgo biloba extract (GBE) possesses protective effects on the glomerulosclerosis of diabetic nephropathy (DN), but its biotransformation in diabetes and DN intestinal bacteria has not yet been recognized. In this work, a validated liquid chromatography-tandem mass spectrometry (LC-MS) method was established for the simultaneous quantification of 14 components in GBE in rat intestinal bacteria matrix, namely ginkgolides A, ginkgolides B, ginkgolides C, bilobalide, rutin, myricetin, quercitrin, quercetin, luteolin, genistein, kaempferol, apigenin, isorhamnetin and genkwanin. Chromatographic separation was performed on a Kromasil-C18 (4.6mm×250mm i.d., 5.0µm) analytical column maintained at 35°C. The mobile phase was a mixture of methanol (A) and 0.1% formic acid in water (B) with a step linear gradient at a flow rate of 1.0mlmin(-1). The calibration curves of these 14 analytes demonstrated good linearity within the test range (R>0.99). This validated method has successfully been applied into the pharmacokinetic study of the 14 components. More importantly, in the pharmacokinetic study, by comparing the time course of the biotransformation by normal, diabetes and DN rat intestinal bacteria, we found that the biotransformation speed and residence time of the 14 compounds in diabetes and DN rats differed obviously from that obtained in normal group, which provided valuable chemical information for further pharmacology and active mechanism research on GBE.

HPLC-DAD and HPLC-ESI-MS analyses of Tiliae flos and its preparations.

In the present study extensive HPLC-DAD, HPLC-ESI-MS and NMR analyses were undertaken in the aqueous preparations (decoctions, infusions) and tinctures of Tilia platyphyllos Scop inflorescences. The aim of this work was to examine in depth the qualitative and quantitative profile of the investigated preparations, which find until today wide applications in pharmaceutical and cosmetic industry, and to propose a validated method for their quality control. An HPLC-DAD-ESI-MS method was developed and optimised for the quantitative determination of the constituents. Marker constituents of Tiliae flos are the flavonoids, while the volatile content is also used for the quality control. However, the analyses of the non-volatile fraction gave complex chromatographic fingerprints containing simple phenolics and low molecular weight procyanidins. The use of different HPLC columns permitted a good separation of the constituents and enabled their quantitation, while HPLC-MS analyses permitted the detection of procyanidin oligomers. Overall, 31 constituents were detected and identified. Extensive preparative chromatographic investigations and 2D-NMR analyses allowed the characterisation of procyanidins as epicatechin derivatives. Finally, the HPLC method was validated and complied with ICH guidelines. This is the first report of detailed analysis of the chemical composition of Tiliae flos.

Matrix effect and optimization of LC-MSn determination of trachylobane-360 in mice blood.

Xylopia langsdorffiana A. St.-Hil. & Tul. (Annonaceae) is popularly known as "pimenteira-da-terra". Various constituents have been isolated from this species, including diterpenes, such as 8(17), 12E, 14-labdatrien-18-oic acid, ent-atisan-7α, 16α-diol (xylodiol), ent-7α-hydroxytrachyloban-18-oic acid (trachylobane-318) and ent-7α-acetoxytrachyloban-18-oic acid, a crystalline solid with a molecular weight of 360 and molecular formula of C22H32O4 (trachylobane-360). When administered intraperitoneally to mice, trachylobane-360 (T-360) significantly inhibits growth of the solid tumor sarcoma 180 transplanted in mice, without causing alterations in biochemical, hematological and histopathological parameters that are frequently associated with the clinical use of antineoplastic. Furthermore, this diterpene blocks voltage-dependent calcium channels (Cav), showing spasmolytic activity. The present study shows that variables such as extraction solvent (methanol, acetonitrile and chloroform), centrifugation force (1000, 7000 and 14,000×g), and centrifugation time (5, 15 and 25min), are important in the liquid-liquid extraction of T-360 from male Swiss mice blood in HPLC-MSn studies. The study confirms matrix influence on recovery and detection of T-360. The recovery for T-360 was 37.02% using chloroform as better extractor solvent, while centrifuged at 14,000×g for 15min demonstrated the importance of the parameters chosen for the extraction/recovery process of analyte. The effect of mice blood matrix for T-360 was -51.23%. This method was optimized by repeating the extraction procedure and acidification of samples. These conditions were essential in increasing recovery (49.47%) by decreasing the matrix effect (-37.60%). The efficiency of the process, after optimization with two extractions and acidification, increased by 14.19% when compared to the initial method, from 18.05% to 32.24%. According to Marchi et al. (2010), the matrix effect does not necessarily need to be reduced or eliminated, but it does need to be identified and quantified. Therefore, these findings are essential for the subsequent evaluation of the pharmacokinetic parameters of this promising natural product.

Ultra-high-pressure liquid chromatography tandem mass spectrometry determination of hallucinogenic drugs in hair of psychedelic plants and mushrooms consumers.

A procedure based on ultra-high-pressure liquid chromatography tandem mass spectrometry has been developed for the determination of mescaline, N,N-dimethyltryptamine, psilocin, psilocybin, salvinorin A in hair of consumers of psychedelic vegetal material such peyote or trichocereus cacti, psilocybe mushrooms, Salvia divinorum or psychedelic beverage ayahuasca. After hair washing with methyl alcohol and diethyl ether and subsequent addition of mescaline-d9 and 3,4-methylenedioxypropylamphetamine as internal standards, hair samples were treated with 250µl VMA-T M3 reagent for 1h at 100°C. After cooling, 100µl M3 extract were diluted with 400µl water and a volume of 10µl was injected into chromatographic system. Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a linear gradient elution with two solvents: 0.3% formic acid in acetonitrile and 5mM ammonium formate pH 3. The mass spectrometer was operated in positive ion mode, using multiple reaction monitoring via positive electrospray ionization. The method was linear from the limit of quantification (0.03-0.05ng/mg depending on analyte under investigation) to 10ng/mg hair, with an intra- and inter-assay imprecision and inaccuracy always less than 15% and an analytical recovery between 79.6% and 97.4%, depending on the considered analyte. Using the validated method, mescaline was found in concentration range of 0.08-0.13ng/mg in hair of peyote smokers, 3.2ng salvinorin A per mg hair were determined in hair from a S. divinorum smoker, 5.6ng N,N-dimethyltryptamine per mg hair from an ayahuasca user and finally 0.8ng psilocybin per ng hair of a psilocybe consumer.

A validated HPLC-ESI-MS/MS method for quantification of 2-hydroxy-4-methoxy benzoic acid from rat plasma and its application to pharmacokinetic study using sparse sampling methodology.

The phenolic compound, 2-hydroxy-4-methoxy benzoic acid (HMBA), is one of the major phytoconstituents of Decalepis arayalpathra (Joseph & Chandra.) Venter, a rare and endemic medicinal plant found in the Western Ghats of India. HMBA has been attributed to possess several biological effects including anti-inflammatory, anti-pyretic, anti-oxidant and anti-diabetic. The present article describes a rapid and sensitive liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) for the determination of HMBA in rat plasma. In brief, the developed assay involves pre-treatment of the plasma samples by an optimized solid phase extraction method (recoveries for HMBA greater than 90%) followed by chromatographic separation on a Cosmosil C18 (150mm×4.6mm i.d.; 5µm particle size) analytical column with mobile phase of methanol and 10mM ammonium formate (95:5 v/v; 0.2% formic acid) delivered at a constant flow rate of 1.0mL/min. The detection and quantification was performed using an Applied Biosystems Hybrid Q-Trap API 2000 mass spectrometer equipped with electrospray ionization source (ESI) functioning in negative mode. The developed assay was validated as per the US FDA bioanalytical guidelines with the calibration curve linear over the concentration range of 5.05-2019.60ng/mL (r(2)≥0.9936) for HMBA from rat plasma. Further, the validated HPLC-MS/MS method was successfully applied to pharmacokinetic study of HMBA after oral administration of D. arayalpathra tuber extracts to female albino Wistar rats using sparse sampling methodology. Following oral administration, the maximum mean concentration in rat plasma (Cmax -1301.57±128.22ng/mL) was achieved at 1.5h (Tmax) and the area under the curve (AUC0-48h) was 8985.02±229.54ngh/mL. The elimination half-life (t1/2) and terminal elimination rate constant (Kel) were 2.48h and 0.28 L/h, respectively.

Formalin-fixed paraffin-embedded tissue as a source for quantitation of carcinogen DNA adducts: aristolochic acid as a prototype carcinogen.

DNA adducts are a measure of internal exposure to genotoxicants. However, the measurement of DNA adducts in molecular epidemiology studies often is precluded by the lack of fresh tissue. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues frequently are accessible, although technical challenges remain in retrieval of high quality DNA suitable for biomonitoring of adducts. Aristolochic acids (AA) are human carcinogens found in Aristolochia plants, some of which have been used in the preparation of traditional Chinese herbal medicines. We previously established a method to measure DNA adducts of AA in FFPE tissue. In this study, we examine additional features of formalin fixation that could impact the quantity and quality of DNA and report on the recovery of AA-DNA adducts in mice exposed to AA. The yield of DNA isolated from tissues fixed with formalin decreased over 1 week; however, the levels of AA-DNA adducts were similar to those in fresh frozen tissue. Moreover, DNA from FFPE tissue served as a template for PCR amplification, yielding sequence data of comparable quality to DNA obtained from fresh frozen tissue. The estimates of AA-DNA adducts measured in freshly frozen tissue and matching FFPE tissue blocks of human kidney stored for 9 years showed good concordance. Thus, DNA isolated from FFPE tissues may be used to biomonitor DNA adducts and to amplify genes used for mutational analysis, providing clues regarding the origin of human cancers for which an environmental cause is suspected.