Reproducible Liquid Chromatography-tandem Mass Spectrometry Research Articles

A validated LC-MS/MS method for determination of antiviral prodrug molnupiravir in human plasma and its application for a pharmacokinetic modeling study in healthy Egyptian volunteers

A fully validated, simple, rapid and reproducible liquid chromatography-tandem mass spectrometry method was developed to determine NHC (N-hydroxycytidine), the active metabolite of Molnupiravir (MOL) in human plasma; one of the limited treatment options for SARS-CoV-2 in plasma of healthy volunteers. The internal standard (IS) used was ribavirin. The extraction of analyte and IS from plasma was performed using acetonitrile as a solvent for protein precipitation. Agilent Zorbax Eclipse plus C18, 4.6 × 150 mm, (5 µm) was used for chromatographic separation using a mixture of methanol0.2 % acetic acid (5:95, v/v) as a mobile phase that was pumped at a flow rate of 0.9 mL/min. Detection was performed on a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) employing positive ESI interface using API4500 triple quadrupole tandem mass spectrometer system, with the transitions set at m/z 260.10 → 128.10 and 245.10 → 113.20 for NHC and IS respectively. Method validation was performed in accordance with United States FDA bioanalytical guidance. The concentration range of 20.0–10000.0 ng/mL was used to establish linearity via weighted linear regression approach (1/x2). Moreover, the analyzed pharmacokinetic data from twelve Egyptian healthy volunteers were used to develop a population pharmacokinetic model for NHC. The developed model was used to perform simulations and evaluate the current MOL dosing recommendations through calculating the maximum concentration (Cmax) “the safety metric” and area under the curve (AUC0-12 h) “the efficacy metric” for 1000 virtual subjects. Geometric mean ratios (GMR) with their associated 90% confidence intervals (CI) compared to literature values were computed. Geometric means of simulation-based Cmax and AUC0-12 were 3827 ng/mL (GMR = 1.05; 90% CI = 0.96–1.15) and 9320 ng.h/mL (GMR = 1.04; 90% CI = 0.97–1.11), respectively indicating that current MOL dosage can achieve the therapeutic targets and dose adjustment may not be required for the Egyptian population. The developed model could be used in the future to refine MOL dosage once further therapeutic targets are identified.

A Pharmacokinetic Study of Ephedrine and Pseudoephedrine after Oral Administration of Ojeok-San by Validated LC-MS/MS Method in Human Plasma.

A sensitive and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was developed and fully validated for the simultaneous determination of ephedrine and pseudoephedrine in human plasma after oral administration of the herbal prescription Ojeok-san (OJS); 2-phenylethylamine was used as the internal standard (IS). Both compounds presented a linear calibration curve (r2 ≥ 0.99) over a concentration range of 0.2–50 ng/mL. The developed method was fully validated in terms of selectivity, lower limit of quantitation, precision, accuracy, recovery, matrix effect, and stability, according to the regulatory guidelines from the U.S. Food and Drug Administration and the Korea Ministry of Food and Drug Safety. This validated method was successfully applied for the pharmacokinetic assessment of ephedrine and pseudoephedrine in 20 healthy Korean volunteers administered OJS.

Development & validation of LC–MS/MS assay for 5-amino-1-methyl quinolinium in rat plasma: Application to pharmacokinetic and oral bioavailability studies

5-Amino-1-methyl quinolinium (5-AMQ) is a potent Nicotinamide N-methyl transferase (NNMT) inhibitor. NNMT is an enzyme that catalyzes the N-methylation of the endogenous substrate nicotinamide, as well as exogenous xenobiotics. NNMT is fundamental to cellular metabolism; NNMT is overexpressed in select tissues (e.g., adipose tissue, skeletal muscle, etc.) in pathophysiological conditions, making it a clinically relevant target for drug development in several chronic diseases including obesity and diabetes. The objective of this study was to develop and validate a simple, sensitive, and reproducible liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the quantification of 5-AMQ in rat plasma and urine samples. 5-AMQ was extracted from plasma and urine by protein precipitation. Chromatographic separation was achieved using an ACE® Excel™ C18 column (2 μm, 50 × 2.1 mm) with a binary gradient solvent system comprising of water (A) and acetonitrile (B) containing 0.1 % formic acid as the mobile phase. Analysis was performed using an API 4000 QTRAP hybrid triple quadruple mass spectrometer and multiple reaction monitoring (MRM) in positive mode at m/z transitions of 159.100 → 90.00 and 162.200 → 117.200 for 5-AMQ and the internal standard, respectively. The standard curves of 5-AMQ in rat urine and plasma samples were linear in the concentration range of 10–2500 ng/mL. The intra-day and inter-day precisions and accuracies for 5-AMQ at four concentration levels in rat plasma and urine samples were found to be within the 15 % FDA acceptance range. Similarly, the accuracy and precision of 5-AMQ quantification in samples diluted up to 20-fold using blank plasma were within the 15 % acceptable range. Furthermore, the extraction recoveries and matrix effects at three concentration levels of rat plasma samples ranged from 99.5 %–110.6 % and -6.1 %–14.1 %, respectively. 5-AMQ was stable in rat plasma samples subjected to standard storage, preparation, and handling conditions, with less than 15 % variation noted at two concentration levels. The validated, sensitive, and reproducible LC–MS/MS method for 5-AMQ in rat plasma and urine samples was effectively applied to a pharmacokinetic study in rats with IV and oral administration of 5-AMQ. 5-AMQ displayed substantial plasma exposures via IV and oral route, with a mean maximum plasma concentration of 2252 ng/mL after oral administration, mean area under the curve (AUC0-∞) of 3708 h.ng/mL and 14431 h.ng/mL for the IV and oral groups, respectively, mean terminal elimination half-life of 3.80 ± 1.10 h and 6.90 ± 1.20 h respectively after intravenous and oral dose, and a good oral bioavailability (F % = 38.4).

Simultaneous LC-MS/MS Determination of Sacubitril, Valsartan and LBQ657 in Human Plasma and Its Application to Clinical Study

A rapid and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine sacubitril, valsartan and a metabolite of sacubitril (LBQ657) in human plasma using sacubitril-d4 and valsartan-d3 as the internal standards. Following protein precipitation, the analytes were operated on an Ultimate® XB-C18 column (2.1 × 50 mm, 3.5 μm, Welch) with a gradient elution with acetonitrile, and 5 mM ammonium acetate and 0.1% formic acid in water as the mobile phase. The detection was performed on a Triple Quad™ 4000 mass spectrometer coupled with an electrospray ionization source (ESI) under positive-ion multiple reaction monitoring mode. The linearities are 2.00-4000, 5.00-10000 and 5.00-10000 ng mL-1 for sacubitril, valsartan and LBQ657, respectively. The accuracy and precision of intra- and inter-day, dilution accuracy, recovery and stability of the method were all within the acceptable limits and no matrix effect or carryover was observed. The suitability of the method was successfully demonstrated in terms of the quantification of sacubitril, valsartan and LBQ657 in plasma samples collected from healthy Chinese volunteers in a clinical trial.

Simple and rapid LC-MS/MS method for determination of sitagliptin in human plasma and application to bioequivalence study

A simple, rapid, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method was developed to determine sitagliptin in human plasma. Diphenhydramine HCl was used as internal standard (IS). The chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 – Fast LC column (100 × 2.1mmID, 2.7) fitted with UHPLC Guard Poroshell 120 EC-C18 (5 × 2.1mmID, 2.7 µm). The mobile phase consisted of 0.1% v/v formic acid and methanol (45:55, v/v) run at a flow rate of 0.45 mL/min at 30 °C. Methanol produced relatively cleaner plasma sample as deproteinization agent. Polytetrafluoroethylene membrane was preferred over nylon membrane as the former produced clear plasma samples. The standard calibration curve was linear over the concentration range of 5–500.03 ng/mL. The within-run precision was 0.53–7.12% and accuracy 87.09–105.05%. The between-run precision was 4.74–11.68% and accuracy 95.02–97.36%. The extended run precision was 3.60–6.88% and accuracy 93.18–95.82%. The recovery of analyte and IS was consistent. Sitagliptin in plasma was stable at benchtop (short term) for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h (post-preparative), after 7 freeze-thaw cycles (−20 ± 10 °C), and 62 days in the freezer (−20 ± 10 °C). Both sitagliptin (analyte) and IS stock solutions were stable for 62 days when kept at room temperature (25 ± 4 °C) and in chiller (2–8 °C). The validated method was successfully applied to a bioequivalence study of two sitagliptin formulations involving 26 healthy Malaysian volunteers.

Quantitative characterization of glutaminolysis in human plasma using liquid chromatography-tandem mass spectrometry.

Glutaminolysis is the metabolic pathway that lyses glutamine to glutamate, alanine, citrate, aspartate, and so on. As partially recruiting reaction steps from the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle, glutaminolysis takes essential place in physiological and pathological situations. We herein developed a sensitive, rapid, and reproducible liquid chromatography-tandem mass spectrometry method to determine the perturbation of glutaminolysis in human plasma by quantifying 13 involved metabolites in a single 20-min run. A pHILIC column with a gradient elution system consisting of acetonitrile-5mM ammonium acetate was used for separation, while an electrospray ionization source (ESI) operated in negative mode with multiple reaction monitoring was employed for detection. The method was fully validated according to FDA's guidelines, and it generally provided good results in terms of linearity (the correlation coefficient no less than 0.9911 within the range of 0.05-800μg/mL), intra- and inter-day precision (less than 18.38%) and accuracy (relative standard deviation between 89.24 and 113.4%), with lower limits of quantification between 0.05 and 10μg/mL. The new analytical approach was successfully applied to analyze the plasma samples from 38 healthy volunteers and 34 patients with type 2 diabetes (T2D). Based on the great sensitivity and comprehensive capacity, the targeted analysis revealed the imperceptible abnormalities in the concentrations of key intermediates, such as iso-citrate and cis-aconitate, thus allowing us to obtain a thorough understanding of glutaminolysis disorder during T2D. Graphical abstract ᅟ.

Sensitive Analysis and Pharmacokinetic Study of Berberrubine Using LC-MS/MS

ObjectiveTo develop a sensitive and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to evaluate the pharmacokinetic behavior of berberrubine (BRB) and its glucuronide (BRBG) in rats. MethodsBRB, BRBG and tetrahydroberberine (THB, internal standard) were isolated by liquid-liquid extraction in rat biological samples. Chromatographic separation was achieved on an Agilent Zorbax Eclipse Plus C18 (2.1 mm × 50 mm, 3.5-Micron) with a gradient mobile phases primarily containing acetonitrile, water with 0.1% formic acid and 5 mm ammonium acetate. The analytes were monitored by MS/MS in positive electrospray ionization mode. Herein, the feasibility of new developed method was validated with respect to specificity, linearity, precision, accuracy, stability, extraction efficiency and matrix effect. The appropriate method was used for the pharmacokinetic study in rats. ResultsThe new developed method could be applied to the pharmacokinetic study of BRB in rats. BRB and BRBG showed good linearity over the ranges of 2-1000 ng/mL and 5-2000 ng/mL, respectively, and precision was no more than 15%. The accuracy, specificity and stability could be acceptable. ConclusionThe new method is sensitive and reproducible. In pharmacokinetic study, BRB showed nonlinear elimination property. Meanwhile, BRB was rapidly absorbed and widely distributed in various tissues with the highest exposure of BRB in kidney and liver. The absolute bioavailability of BRB was determined to be 8.2% and at the dose of 40 mg/kg, a total of 44% BRB was excreted in urine and feces.

Simultaneous analysis of allopurinol and oxypurinol using a validated liquid chromatography–tandem mass spectrometry method in human plasma

The present study describes a simple, reliable and reproducible liquid chromatography–tandem mass spectrometry method (LC–MS/MS) for the simultaneous determination of allopurinol and its active metabolite, oxypurinol in human plasma for a pharmacokinetic/bioequivalence study. After protein precipitation (PPT) of 100µL plasma sample with 1.0% formic acid in acetonitrile, the recovery of the analytes and allopurinol-d2 as an internal standard ranged from 85.36% to 91.20%. The analytes were separated on Hypersil Gold (150mm×4.6mm, 5µm) column using 0.1% formic acid-acetonitrile (98:2, v/v) as the mobile phase. Quantification was done using electrospray ionization in the positive mode. The calibration concentration range was established from 60.0 to 6000ng/mL for allopurinol and 80.0–8000ng/mL for oxypurinol. Matrix effect in human plasma, expressed as IS-normalized matrix factors ranged from 1.003 to 1.030 for both the analytes. The developed method was found suitable for a clinical study with 300mg allopurinol tablet formulation in healthy subjects.

An LC-MS/MS method for determination of novel fungicide pyraoxystrobin in rat plasma and tissues: Toxicokinetics and tissue distribution study

A simple, specific and reproducible liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed for the determination of pyraoxystrobin in rat plasma and tissues. Chromatographic separation was achieved on a Zorbax Extend-C18 column (50×2.1mm I. D., 3.5µm), using a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid (v/v) at a flow rate of 0.5mLmin−1. Pyraoxystrobin and picoxystrobin (internal standard) were detected without interference in the selected reaction monitoring (SRM) mode with positive electrospray ionization. Further, the method was validated following FDA guideline. The calibration curves for plasma and tissues were linear over a concentration range of 1.00–200ngmL−1, with lower limits of quantitation of 1.00ngmL−1. Mean extraction recoveries in plasma and tissues ranged from 101.4% to 108.2% and from 49.1% to 59.4%, respectively. The intra-day and inter-day precision in plasma and tissues were within 9.9% and 8.9%, and the intra-day and inter-day accuracy ranged from 88.7% to 110.7% and 93.2% to 108.7%, respectively. Finally, the validated method was successfully applied to toxicokinetics and tissue distribution studies after oral administration of pyraoxystrobin to rats.

Sensitive and accurate liquid chromatography–tandem mass spectrometry methods for quantitative determination of a novel hepatitis C NS5B inhibitor BMS-791325 and its active metabolite in human plasma and urine

BMS-791325 is a novel hepatitis C NS5B inhibitor which is currently in clinical development. To support pharmacokinetic (PK) assessments, sensitive, accurate, precise, and reproducible liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods have been developed and validated for the quantitation of BMS-791325 and its active N-demethyl metabolite (BMS-794712) in human plasma and urine. Plasma and urine samples were extracted with methyl-t-butyl ether followed by an LC–MS/MS analysis which was conducted in a multiple reaction monitoring (MRM) mode for the simultaneous detection of the two analytes in human plasma (0.1–50ng/mL) and in human urine (5–2500ng/mL). Intra-run precision (3.0% R.S.D.), inter-run precision (5.3% R.S.D.), and accuracy (±4.7% deviation) from plasma and urine quality control samples provide evidence of the methods accuracy and precision. Selectivity, stability in matrices, extraction recovery, matrix effect on LC–MS detection, and interference of coadministered drugs (famotidine and ritonavir) were all acceptable. Reproducibility of the plasma method was demonstrated by reanalysis of a portion of study samples. The results of cross-validations demonstrated the equivalency of two methods validated in two labs. The plasma method was applied to the analysis of several thousand clinical study samples for PK evaluations of the drug in normal healthy subjects and in patients. The urine method was used in the first in human study to evaluate renal clearance and urinary recovery.

Bioequivalance and pharmacokinetic study of febuxostat in human plasma by using LC-MS/MS with liquid liquid extraction method.

A bioequivalence study was proved of generic Febuxostat 80 mg tablets (T) in healthy volunteers.For this purpose, Authors developed a simple, sensitive, selective, rapid, rugged and reproducible liquid chromatography–tandem mass spectrometry method for the quantification of Febuxostat (FB) in human plasma using Febuxostat D7 (FBD7) as an internal standard (IS) was used. Chromatographic separation was performed on Ascentis Express C18 (50x4.6 mm, 3.5 μ) column. Mobile phase composed of 10 mM Ammonium formate: Acetonitrile (20:80 v/v), with 0.8 mL/min flow-rate. Drug and IS were extracted by Liquid- liquid extraction. FB and FBD7 were detected with proton adducts at m/z 317.1→261.1 and 324.2→262.1 in multiple reaction monitoring (MRM) positive mode respectively. The method was validated with the correlation coefficients of (r2) ≥ 0.9850 over a linear concentration range of 1.00-8000.00 ng/mL. This method demonstrated intra and inter-day precision within 2.64 to 3.88 and 2.76 to 8.44% and accuracy within 97.33 to 99.05 and 100.30 to 103.19% for FB. This method is successfully applied in the Bioequivalence study of 9 human volunteers.

Quantitative analysis of eletriptan in human plasma by HPLC-MS/MS and its application to pharmacokinetic study

Authors developed a simple, sensitive, selective, rapid, rugged, and reproducible liquid chromatography-tandem mass spectrometry method for the quantification of eletriptan (EP) in human plasma using naratriptan (NP) as an internal standard (IS). Chromatographic separation was performed on Ascentis Express C18, 50 × 4.6 mm, 2.7 μm column. Mobile phase was composed of 0.1% formic acid: methanol (40:60 v/v), with 0.5 mL/min flow rate. Drug and IS were extracted by liquid-liquid extraction. EP and NP were detected with proton adducts at m/z 383.2→84.3 and 336.2→97.8 in multiple reaction monitoring (MRM) positive mode, respectively. The method was validated with the correlation coefficients of (r(2)) ≥ 0.9963 over a linear concentration range of 0.5-250.0 ng/mL. This method demonstrated intra- and inter-day precision within 1.4-9.2% and 4.4-5.5% and accuracy within 96.8-103% and 98.5-99.8% for EP. This method is successfully applied in the bioequivalence study of 24 human volunteers.

Microanalysis of the antiretroviral nevirapine in human hair from HIV-infected patients by liquid chromatography-tandem mass spectrometry

Sufficient drug exposure is crucial for maintaining durable responses to HIV treatments. However, monitoring drug exposure using single blood samples only provides short-term information and is highly subject to intra-individual pharmacokinetic variation. Drugs can accumulate in hair over a long period of time, so hair drug levels can provide drug exposure information over prolonged periods. We now report on a specific, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method for measuring nevirapine (NVP), a widely used antiretroviral drug, levels in human hair using even a single short strand of hair. Hair samples are cut into small segments, and the drug is extracted in methanol/trifluoroacetic acid (v/v, 9:1) shaken at 37 °C in a water bath overnight, followed by liquid-liquid extraction under alkaline conditions. The extracted samples are then separated on a BDS-C(18) column with a mobile phase composed of 50% acetonitrile containing 0.15% acetic acid and 4 mM ammonium acetate with an isocratic elution for a total run time of 3 min and detected by triple quadrupole electrospray multiple reaction mode at precursor/product ion at 267.0 > 225.9 m/z. Deuterated nevirapine-d5 was used as an internal standard. This method was validated from 0.25 to 100 ng/mg using 2 mg hair samples. The accuracies for spiked NVP hair control samples were 98-106% with coefficients of variation (CV) less than 10%. The CV for incurred hair control samples was less than 7%. The extraction efficiency for incurred control hair samples was estimated at more than 95% by repeated extractions. This method has been successfully applied to analyze more than 1,000 hair samples from participants in a large ongoing cohort study of HIV-infected participants. We also showed that NVP in human hair can easily be detected in a single short strand of hair. This method will allow us to identify drug non-adherence using even a single strand of hair.

Application of a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method to the pharmacokinetics of ON01910 in brain tumor-bearing mice

ON01910 is a small molecular weight benzyl styryl sulfone currently under investigation as a novel anticancer agent. The purpose of the investigation was to develop a sensitive and reproducible liquid chromatography–tandem mass spectrometry (LC/MS/MS) method to quantitate levels of ON01910 in small amounts of five biological matrices; mouse plasma, feces, urine, normal brain and brain tumor. For all matrices, protein precipitation sample preparation was used that led to linear calibration curves with coefficients of determination greater than 0.99. The lower limit of quantitation (LLOQ) for all matrices was 5 ng/ml except that for mouse urine which was 10 ng/ml. The calibration standard curves were reproducible for all matrices with inter- and intra-day variability in precision and accuracy being less than 15% at all quality control concentrations except for the LLOQ in mouse plasma for which the accuracy was within 17%. The assay was successfully applied to characterize the systemic pharmacokinetics of ON01910 as well as its disposition in brain and brain tumor in mice. ON01910 exhibited a clearance of 3.61 ± 0.85 l/h/kg and a half life of 8.66 ± 3.30 h at 50 mg/kg dose given I.V.

An improved LC‐MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and its active metabolites in human plasma and its application to a pharmacokinetic study in patients

In this study, a sensitive, selective and reproducible liquid chromatography-tandem mass spectrometry method for the simultaneous determination of 1,5-dicaffeoylquinic acid (1,5-DCQA) and its active metabolites, 1-caffeoyl-5-feruoylquinic acid and 1,5-O-diferuoylquinic acid, in human plasma, using puerarin as internal standard, was developed and validated. Analytes were extracted from plasma samples by liquid-liquid extraction with ethyl acetate, separated on a C(18) reversed-phase column with water containing 5 mM ammonium acetate and acetonitrile as the mobile phase and detected by electrospray ionization mass spectrometry in negative selected reaction monitoring mode. The accuracy and precision of the method were acceptable and linearity was good over the range 1-200 ng/mL for each analyte. In addition, the selectivity, extraction recovery and matrix effect were satisfactory too. The validated LC-MS/MS method was successfully applied to phase II clinical pharmacokinetic study of 1,5-DCQA in patients.

Determination of Olanzapine in Whole Blood Using Simple Protein Precipitation and Liquid Chromatography-Tandem Mass Spectrometry

A simple, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of the antipsychotic drug olanzapine in whole blood using dibenzepine as internal standard (IS). After acidic methanol-induced protein precipitation of the whole blood samples, olanzapine and IS were chromatographed on a reversed-phase Zorbax Extend-C(18)-column at pH 9.0. Quantification was performed on a triple-quadrupole mass spectrometer employing electrospray ionization technique operating in multiple reaction monitoring and positive ion mode. Total chromatographic run time was 15 min, and calibration curve was linear over the concentration range of 0.005 to 0.50 mg/kg olanzapine in whole blood. The method was validated for selectivity, matrix interference, recovery, linearity, limit of detection, limit of quantitation (LOQ), accuracy, precision, and stability. The absolute recovery obtained was 103% for olanzapine and 68% for IS. An LOQ of 0.005 mg/kg olanzapine in whole blood was achieved. Inter- and intraday precision were less than 11% within concentrations from 0.01 to 0.50 mg/kg, and the accuracy ranged from 85 to 115%. The method was subsequently applied to 27 authentic samples, of which 20 were postmortem blood samples, from forensic investigations.

LC–MS–MS Development and Validation for Simultaneous Quantitation of Metformin, Glimepiride and Pioglitazone in Human Plasma and Its Application to a Bioequivalence Study

A simple, precise and reproducible liquid chromatography–tandem mass spectrometry method has been developed and validated according to the Food and Drug Administration guidelines for the simultaneous quantitation of antidiabetic drugs metformin, glimiperide and pioglitazone in human plasma using glipizide as an internal standard. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring and positive ion mode. Inter-batch and intra-batch coefficient of variation across four validation runs for the quality control samples was less than 7%. The accuracy determined at quality control levels was within 92.81–105.13%. The method was applied to a bioequivalence study.

Abstract B126: Does adult soy intake influence total estrogen levels and estrogen metabolism patterns?

Abstract B126 Background Soy foods are a dietary staple in Asian countries and may play a role in this region’s historically low rates of breast cancer incidence. Isoflavones found in soy have structural similarities to estrogens and could exert protective effects by interacting with estrogen receptors, binding proteins, or metabolic enzymes. In a sample of Asian-American women we test whether soy is associated with urinary concentrations of estrogens and estrogen metabolites (EM). Methods This analysis was conducted among controls from a population-based case-control study of breast cancer among women of Chinese, Japanese and Filipino ancestry, aged 20-55 years, and living in San Francisco-Oakland (CA), Los Angeles (CA) and Oahu (HI). Of 966 controls, we included the 571 (59%) women who donated a 12-hour urine and had not been pregnant or lactating or used exogenous hormones in the last 6 months. At urine collection, 168 women were postmenopausal, 233 were pre-menopausal in mid-luteal phase, and 170 were in other categories. Fifteen EM were measured simultaneously with a highly sensitive and reproducible liquid chromatography-tandem mass spectrometry method. Participants had been queried about their usual adult frequency of intake of tofu and other selected soy foods. Robust regression was used to model the associations of soy intake (in tertiles) with EM measures. Models were adjusted for age, ethnicity, body mass index (BMI), and birthplace (Asia/West). Results No statistically significant associations were seen between soy intake and urinary concentrations of estrone, estradiol, estriol, or total EM in postmenopausal women or premenopausal women. In postmenopausal but not premenopausal women, we found significant trends across tertiles of soy intake for EM grouped by metabolic pathways. The 2- and 4-hydroxylated EM and the methylated catechols, each expressed as a percent of total EM, increased with soy intake, while relative levels of EM in the 16-hydroxylation pathway decreased. Models adjusted for age and ethnicity were similar. Factor analysis was used to confirm EM groupings. Among postmenopausal women, factor analysis identified four independent EM patterns, termed ‘catechols’, ‘methylated catechols’, ‘16-hydroxylation pathway EM’, and ‘parent estrogens’ based on the predominant EM. Factors for premenopausal women were similar. Discussion Our findings do not support the hypothesis that soy intake influences endogenous production of estrogens but they do suggest that soy intake can modify estrogen metabolism in postmenopausal women. Increased ratios of 2-hydroxyestrone to 16α-hydroxyestrone have been associated with lower breast cancer risk in some epidemiologic studies. Methylation of catechol estrogens results in deactivation of these metabolites and prevents conversion into genotoxic quinones. Thus, the trends by soy intake represent variations in EM metabolism that could reduce breast cancer risk. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B126.

LC–Tandem-MS Validation for the Quantitative Analysis of Levonorgestrel in Human Plasma

In this study, a rapid, sensitive and reproducible liquid chromatography–tandem mass spectrometry method for the determination of levonorgestrel in human plasma, was developed and validated. With a structural analogue norethindrone as the internal standard, levonorgestrel was extracted from plasma using ethyl acetate. The organic layer was evaporated to dryness and the residue was reconstituted in mobile phase. An aliquot of 20 μL was chromatographically analyzed on a Phenomenex Luna C18 column with water and acetonitrile as the mobile phase. Selected reaction monitoring was specific for mass detection employing positive electrospray ionization. The calibration standards were linear over the concentration range 0.625–40 ng mL−1. The intra- and inter-day precision over the entire concentration range were less than 8.16%. The method was found to be suitable for application to a pharmacokinetic study after oral administration of 1.5 mg levonorgestrel tablet to 20 healthy female volunteers.

Preparative chromatographic purification of diastereomers of silybin and their quantification in human plasma by liquid chromatography–tandem mass spectrometry

A novel preparative HPLC method separating silybin has been developed to meet the need for both silybin A and silybin B standard. After the preparation of silybin A and silybin B standard, a simple, sensitive, selective and reproducible liquid chromatography–tandem mass spectrometry (LC–MS–MS) method with negative electrospray ionization (ESI) was developed for the quantification of silybin A and silybin B in human plasma. Following rapid sample preparation, silybin A, silybin B and naringin (internal standard, ISTD) were separated on a Zorbax Eclipse XDB-C 18 column, using methanol–water containing 0.1% formic acid (48:52, v/v) as the mobile phase. The mass spectrometer was operated in selected reaction monitoring (SRM) mode using the transition m/ z 481.1 → 300.9 for both silybin A and silybin B and m/ z 579.2 → 271.1 for naringin, respectively. Linear calibration curves were obtained in the concentration range of 2–5000 ng/ml with a lower limit of quantitation (LLOQ) of 2 ng/ml for both silybin A and silybin B, respectively. The intra- and inter-day precision values were below 7.5% and accuracy was within ±4.9% at all three quality control (QC) levels, for both silybin A and silybin B, respectively. This method was successfully applied to the stereospecific analysis of silybin in plasma samples from a pharmacokinetic study of silybin A and silybin B in 22 healthy male Chinese volunteers after a single oral dose of silybin–phosphatidylcholine complex (equivalent to 280 mg silybin, including 133 mg silybin A and 147 mg silybin B).