Blank Human Plasma Research Articles

Expedient methodology for total methotrexate polyglutamation pool determination in human erythrocytes

The measurement of methotrexate polyglutamate metabolites in red blood cells has potential to aid in individualization of methotrexate therapy in rheumatoid arthritis and juvenile idiopathic arthritis. In this report a method is presented for rapid analysis of these metabolites in human red blood cells. The analytical procedure is a simple “one pot” pre-column reaction involving the addition of sodium dithionite as a reducing agent followed by 15 minutes of boiling. After centrifugation the supernatant is introduced into a conventional HPLC system equipped with a fluorescence detector, without the need for further workup. By performing the derivatization reaction pre-column, a time consuming (6–14 h) commonly used deglutamation procedure utilizing blank human plasma, becomes obsolete. Using the described procedure the total sample preparation time for a 50 sample run should not exceed 1–1.5 hours. The chromatographic run time per sample is 7 minutes using isocratic elution conditions. The method was found to be linear over the clinical relevant concentration range of 10–500 nM of intra-cellular methotrexate polyglutamates. The intra-run mean accuracy of the target value was between 98.1% and 106.0%. The intra-run precision was between 1.2% and 8.8%.

Imatinib assay by HPLC with photodiode-array UV detection in plasma from patients with chronic myeloid leukemia: Comparison with LC-MS/MS

Background Imatinib, a competitive inhibitor of BCR–ABL tyrosine kinase, is now the first-line treatment for chronic myelogenous leukemia (CML). Therapeutic drug monitoring targeting trough plasma levels of about 1000 ng/mL may help to optimize the therapeutic effect. Methods We developed a high-performance liquid chromatography (HPLC) method with UV/Diode Array Detection (DAD) for trough imatinib concentration determination in human plasma. Imatinib trough levels were measured in plasma from 65 CML patients using our method and LC-MS/MS as the reference method. Results with these two methods were compared using Deming regression, chi-square test, and sign test. Results The calibration curve was prepared in blank human plasma. HPLC-UV/DAD calibration curves were linear from 80 to 4000 ng/mL, and the limit of quantification was set at 80 ng/mL. The between-day variation was 6.1% with greater than 96% recovery after direct plasma deproteinization and greater than 98% recovery from the column. No significant differences in imatinib plasma levels were found between HPLC-UV/DAD and LC-MS/MS. Conclusions This HPLC-UV/DAD method was sufficiently specific and sensitive for imatinib TDM, with no evidence of interference. Our rapid inexpensive HPLC-UV/DAD method that requires only widely available equipment performs well for plasma imatinib assays.

Quantitative determination of clopidogrel active metabolite in human plasma by LC–MS/MS

A quantitative method for the determination of clopidogrel active metabolite (AM) in human plasma was developed and validated using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Clopidogrel AM contains a thiol group, thus requiring stabilization in biological samples. The alkylating reagent 2-bromo-3′-methoxyacetophenone was used to stabilize clopidogrel AM in blood. An analog of the derivatized clopidogrel AM was used as the internal standard (IS). The derivatized samples were subjected to solid-phase extraction with a C2 disk plate and the overall procedure exhibited good reaction (more than 90%) and recovery efficiencies (from 85% to 105%). The derivative of clopidogrel AM (MP-AM) and IS were separated on an ODS column and quantified by tandem mass spectrometry with electrospray ionization. No significant endogenous peaks corresponding to MP-AM or IS were detected in blank human plasma samples, and no significant matrix effect was observed for MP-AM and IS in human plasma samples (from 102% to 121%). The calibration curve ranged from 0.5 to 250 ng/mL with good linearity, and extended by validation of a 50-fold dilution. In the intra- and inter-assay reproducibility tests, the accuracy and precision were within 12% relative error and 6% coefficient of variation, respectively. The derivatized MP-AM was stable in human plasma for 4 months at −80 °C. The validated method was successfully used to analyze clinical samples and determine the pharmacokinetics of clopidogrel AM.

Liquid chromatography-electrospray ionization-tandem mass spectrometry for simultaneous analysis of chlorogenic acids and their metabolites in human plasma

A method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was developed for the simultaneous analysis of nine chlorogenic acids (CGAs), three isomers each of caffeoylquinic acids (CQAs), feruloylquinic acids (FQAs) and dicaffeoylquinic acids (dCQAs), and their two metabolites, caffeic acid (CA) and ferulic acid (FA), in human plasma. In simultaneous multiple reaction monitoring (MRM) measurements using ESI-MS/MS with a negative ion mode, a deprotonated molecular ion derived from each of the 11 molecules was used as a precursor ion while three diagnostic product ions characteristic for each were selected for the qualitative analysis. To obtain maximal intensities for all diagnostic product ions, the collision energy was optimized for each one. LC separation was achieved under conditions of a reversed-phase Inertsil ODS-2 column combined with a gradient elution system using 50 mM acetic acid with 3% acetonitrile aqueous solution and 50 mM acetic acid with 100% acetonitrile. In the quantitative analysis, one of the three diagnostic product ions for each of the 11 molecules was selected. Application of simultaneous LC-ESI-MS/MS MRM measurements to analyze the 11 standards spiked into blank human plasma indicated that all diagnostic product ions were detected without any interference, and that the sensitivity, linearity and recovery of this method were acceptable. When using this method to analyze those 11 molecules in the plasma after oral ingestion of 250 ml of a drink containing a green coffee bean extract (300 mg CGAs), all 11 molecules were identified and CQAs, FQAs and FA were quantified. CQAs, FQAs and dCQAs in human plasma were detected for the first time. This method should be useful to understand the biological and pharmacological effects of CGAs, such as improvement of human hypertension.

Determination of imatinib mesylate and its main metabolite (CGP74588) in human plasma and murine specimens by ion‐pairing reversed‐phase high‐performance liquid chromatography

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of imatinib, a tyrosine kinase inhibitor, and its main metabolite N-desmethyl-imatinib (CGP74588) in human plasma and relevant murine biological matrices. A simple HPLC assay for the individual quantification of imatinib and CGP74588 in murine specimens has not been reported to date. Sample pre-treatment involved liquid-liquid extraction with tert-butyl-methyl ether. Imatinib, CGP74588 (metabolite) and the internal standard 4-hydroxybenzophenone were separated using a narrow bore (2.1 x 150 mm) stainless steel Symmetry C(18) column and detected by UV at 265 nm. The mobile phase consisted of 28% (v/v) acetonitrile in 50 mM ammonium acetate buffer pH 6.8 containing 0.005 M 1-octane sulfonic acid and was delivered at 0.2 mL/min. The calibration curve was prepared in blank human plasma and was linear over the dynamic range 10 ng/mL to 10 microg/mL). The accuracy was close to 100% and the within-day and between-day precisions were within the generally accepted 15% range. The validation results showed that the assay was selective and reproducible. This method was applied to study the pharmacokinetics of imatinib and its main metabolite in human and mice.

Development and validation of a method for quantitative determination of valsartan in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of valsartan in human plasma was developed and validated. A 0.5 ml aliquot was extracted using solid-phase extraction in an Empore ® high performance extraction disk plate, universal resin 96-well format. The estimated calibration range of the method was 2–2000 ng/ml. The method was fully validated with intra-day mean accuracy and precision of 94.8–107% and 2.19–5.40% and inter-day mean accuracy and precision of 93.5–105% and 1.87–5.67%, respectively. No significant loss of valsartan in processed samples was confirmed in processed samples for up to 24 h at 10 °C. Sample dilution up to 50-fold with blank human plasma provided acceptable analyses. No interference peaks or matrix effects were observed. No effect of QC sample location results was observed in a 96-well plate. This LC-MS/MS technique was found to improve quantitative determination of valsartan allowing its pharmacokinetic evaluation with clinically relevant doses.

Development and validation for high selective quantitative determination of metformin in human plasma by cation exchanging with normal-phase LC/MS/MS

An assay based on cation exchange solid-phase extraction and liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed for the quantitative determination of metformin in human plasma. The analytical method consists of cation exchange solid-phase extraction (VersaPlate CBA) without any further evaporation/dissolution steps and cation exchange-based HPLC separation (Capcell Pak SCX column) with a normal-phase gradient system followed by semi-micro LC/MS/MS in positive ion selected reaction monitoring mode using electrospray ionization. The method exhibited excellent performance in terms of selectivity, robustness, short run time (7 min/sample) and simplicity of sample preparation. The calibration range was 10–1000 ng/ml with 0.2 ml of plasma. Intra- and inter-day mean accuracies were within the ranges of 100.3–105.0% and 101.2–105.3%, respectively. Intra- and inter-day precisions were within the ranges of 0.8–1.9% and 1.5–8.6%, respectively. Mean absolute recovery was 67.0% for metformin. No apparent loss of metformin after extraction was observed in an autosampler at 10 °C for 24 h. Dilution of metformin by blank human plasma up to 20-fold was tested and revealed no impact on the results of determination. Furthermore, the method exhibited high selectivity, since no effect on metformin analysis was observed on comparison of samples with or without nateglinide and other agents in plasma. Results obtained with the method were also comparable to a published LC–UV method on cross-validation. This method can be applied to various clinical pharmacokinetic studies of metformin.

Simultaneous determination of ginsenoside Rb 1 and Rg 1 in human plasma by liquid chromatography–mass spectrometry

A liquid chromatographic–mass spectrometric (LC/MS) method for the simultaneous determination of ginsenoside Rb 1 and Rg 1 in human plasma was developed. The method involved the protein precipitation followed by analysis of ginsenoside Rb 1 and Rg 1 in an Atlantis C 18 column with the gradient elution of acetonitrile and ammonium formate (10 mM, pH 3.0) at a flow rate of 0.2 ml/min. The analytes were determined using electrospray negative ionization mass spectrometry in the selected ion monitoring mode. The standard curves for ginsenoside Rb 1 and Rg 1 were linear over the concentration range of 10.0–1000 ng/ml. The lower limit of quantification was 10.0 ng/ml using 100 μl plasma sample. The coefficient of variation of intra- and inter-day assays for ginsenoside Rb 1 and Rg 1 at three quality control levels ranged from 1.0 to 6.8% and 5.4 to 9.8%, respectively. Ginsenoside Rb 1 and Rg 1 were stable in blank human plasma at room temperature for 24 h and following three freeze–thaw cycles.

High Performace Liquid Chromtographic Determination of Nicardipine Hydrochloride in Human Plasma

A sensitive high‐performance liquid chromatographic method was developed for the estimation of nicardipine hydrochloride in human plasma. Varying amount of nicardipine hydrochloride (2.5 to 150 ng/0.5 mL) and fixed quantity (100 ng/0.5 mL) of nifedipine (internal standard) was added to blank human plasma, and a single step extraction was carried out with ethyl acetate. The mixture was centrifuged, ethyl acetate layer separated, dried and reconstituted with 100 μL of acetonitrile. Twenty microliters of this solution was injected into a reverse phase C‐18 column using a mobile phase consisting of acetonitrile: 0.02 M potassium dihydrogen phosphate (pH 4.0) in the ratio of 60:40 v/v and the eluents were monitored at 239 nm. The method was validated for its linearity, precision and accuracy. The calibration curve was linear in the range of 5‐150 ng/0.5 mL of plasma and the lower detection limit was 2.5 ng/0.5 mL of plasma. The intra‐ and inter‐day variation was found to be less than 2.5% indicating that the method is highly precise. The mean recovery of nicardipine hydrochloride from plasma samples was 89.6±2.60%. The proposed HPLC method was applied for the estimation of nicardipine hydrochloride in human plasma after oral administration of an immediate release nicardipine hydrochloride capsule (dose 30 mg) to 6 adult male volunteers. There was no interference of either the drug metabolites or other plasma components with the proposed HPLC method for the estimation of nicardipine hydrochloride in human plasma. Due to its simplicity, sensitivity, high precision and accuracy, the proposed HPLC method may be used for biopharmaceutical and pharmacokinetic evaluation of nicardipine hydrochloride and its formulations in humans

Liquid chromatography-tandem mass spectrometry for the determination of a new oxazolidinone antibiotic DA-7867 in human plasma.

A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of a new ox-azolidinone antibiotic DA-7867, (S)-[N-3-(4-(2-(1-methyl-5-tetrazolyl)-pyridine-5-yl)-3- fl uorophenyl)-2-oxo-5-oxazolidinyl]methyl acetamide, in human plasma was developed. DA-7867 and internal standard, linezolid, were extracted from human plasma with ethyl acetate at acidic pH. A reverse-phase LC separation was performed on Luna C(8) column with the mixture of acetonitrile-ammonium formate (10 mm, pH 4.5; 35:65, v/v) as mobile phase. The analytes were determined using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The lower limits of quanti fi cation for DA-7867 was 2.5 ng/mL. The single liquid-liquid extraction quantitatively recovered DA-7867 and internal standard from plasma samples at the ranges of 82.2-86.7%. DA-7867 was stable in blank human plasma at room temperature for 24 h and following three freeze-thaw cycles.

Bioanalysis of zosuquidar trihydrochloride (LY335979) in small volumes of human and murine plasma by ion−pairing reversed-phase high-performance liquid chromatography

We have developed and validated a sensitive and selective method for the quantitative determination of the P-glycoprotein inhibitor zosuquidar (LY335979) in human and murine plasma using only 50 μl sample volumes. Sample pretreatment involved liquid-liquid extraction with tert-butyl methyl ether. Zosuquidar and the internal standard chlorpromazine were separated using a narrow bore column ( 2.1 mm×150 mm) packed with 3.5 μm symmetry C 18 material. The mobile phase consisted of 38% (v/v) acetonitrile in 50 mM ammonium acetate buffer pH 3.8 containing 0.005 M 1-octyl sulfonic acid and was delivered at 0.2 ml/min. Detection was performed with a fluorescence detector set at an excitation wavelength of 260 nm and an emission wavelength of 460 nm. The calibration curve was prepared in blank human plasma and was linear over the dynamic range (10–1000 ng/ml). The lower limit of quantitation was 20 ng/ml. The validation results showed that the assay was selective and reproducible. Within the range of the calibration curve the accuracy was close to 100% and within-day and between-day precision were within the generally accepted 15% range. This method was applied to study the pharmacokinetics of i.v. administered zosuquidar in mice. The sensitivity of the assay was sufficient to determine the drug concentration in plasma samples obtained up to 24 h after administration.

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of Aplidin, a novel marine-derived antineoplastic agent, in human plasma.

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel marine-derived depsipeptide, Aplidin, in human plasma. The method was validated to demonstrate the specificity, recovery, limit of quantitation (LOQ), accuracy, and precision of measurements. The calibration range for Aplidin was established using Aplidin standards from 0.05-50 ng/mL in blank human plasma. The multiple reaction monitoring, based on the transition m/z 1110.7 --> 295.3, was specific for Aplidin, and that based on the transition m/z 1112.6 --> 297.3 was specific for didemnin B (the internal standard); no endogenous materials interfered with the analysis of Aplidin and didemnin B from blank human plasma. The assay was linear over the concentration range 0.05-50.0 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9979 to 0.9999. The mean intra- and interday accuracies for all calibration standards (n = 12) ranged from 97 to 106% (</=6% bias), and the mean interday precision for all calibration standards was less than 8.3%. The mean intra- and interday assay accuracy for all quality control replicates (n = 12), determined at each QC level throughout the validated runs, remained below 12 and 7%, respectively. The mean intra- and interday assay precision was less than 13.1 and 10.7% for all QC levels, respectively. The assay is currently used to measure Aplidin plasma concentrations to support clinical trials.

LC-MS-MS determination of nemorubicin (methoxymorpholinyldoxorubicin, PNU-152243A) and its 13-OH metabolite (PNU-155051A) in human plasma

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for quantitative determination of nemorubicin, (PNU-152243A, 3′-deamino-3′[2( S)-methoxy-4-morpholinyl]doxorubicin) hydrocloride and its reduced metabolite PNU-155051 in human plasma has been developed and validated. The method involved solid phase extraction (SPE) in 96-well plates. Plasma samples (0.5 ml plasma, spiked with doxorubicin as internal standard and diluted with 0.5 ml of 0.01 M borate buffer, pH 8.4) were extracted using Oasis™ HLB SPE material. The elution of PNU-152243, PNU-155051 and of IS was performed with 1 ml of methanol:0.1 M formic acid mixture (90:10, v/v). The organic phase was reduced to dryness under a stream of nitrogen at 20 °C and the residue was reconstituted with 0.25 ml of 10 mM ammonium formate buffer pH 4.15:acetonitrile mixture (90:10, v/v). Aliquots of 60 μl of the resulting solution were injected onto the LC-MS-MS system. A Zorbax SB C18 column (2.1×150 mm, 3.5 μm) was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 10 mM pH 4.15:acetonitrile (73:27, v/v) with a flow-rate of 0.2 ml/min. Detection was achieved by a PE-SCIEX API 3000 with Turbo IonSpray interface, and multiple reaction monitoring (645→321 for PNU-152243, 647→363 for PNU-155051 and 545→345 m/ z for doxorubicin) operated in positive ion mode. A weighted linear regression was used to calculate PNU-152243 and PNU-155051 concentrations in QC and unknown samples. Linearity, precision, accuracy and recovery of the method were evaluated over the concentration range of 0.1–5 ng/ml for both compounds. No interference from blank human plasma was observed. The suitability of the method for in vivo samples was assessed by the analysis of samples obtained from patients who had received a single intrahepatic artery dose of PNU-152243A.

Determination of cyclosporin A in human and mouse plasma by reversed-phase high-performance liquid chromatography

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 227 nm has been validated for the determination of cyclosporin A in human and mouse plasma. The cyclosporin D analog PSC 833 was used as internal standard. Plasma samples were pretreated by liquid–liquid extraction with diethyl ether. A good chromatographic separation between cyclosporin A, the internal standard and two potentially interfering endogenous peaks was achieved using a stainless steel column packed with 5 μm Nova-Pak phenyl material operated at 72°C, and a mobile phase consisting of acetonitrile–methanol–water (20:52:28, v/v/v). The calibration curve for cyclosporin A in human plasma was linear over the tested concentration range of 0.11 to 5.34 μ M. Murine plasma samples (200 μl) were diluted up to a total volume of 500 μl with blank human plasma and the concentrations were read from the calibration curve prepared in human plasma. The lower limit of quantitation was 0.11 μ M using 500 μl of human plasma and 0.28 μ M using 200 μl of mouse plasma. The validation data showed that the assay is sensitive, selective and reproducible for determination of cyclosporin A. The applicability was demonstrated in a pharmacokinetic experiment where mice received oral cyclosporin A.

Determination of the covalent adducts of the novel anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid in biological samples by high-performance liquid chromatography

The reversed-phase HPLC methods were developed to determinate the covalently bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by alkaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 μl of the mixture was injected onto a Spherex C 18 column (150×4.6 mm; 5 μm) at a flow-rate of 2.5 ml/min. The mobile phase comprising of acetonitrile:10 m M ammonium acetate buffer (24:76, v/v, pH 5.8) was used in an isocratic condition, and DMXAA was detected by fluorescence. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed in the concentration range of 0.5–40 μ M in washed blank human plasma or HSA prior to alkaline hydrolysis. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The limit of detection for the covalent adduct in human plasma or HSA is 0.20 μ M. The methods presented good accuracy, precision and sensitivity for use in the preclinical and clinical studies.

Determination of MAG-Camptothecin, a new polymer-bound Camptothecin derivative, and free Camptothecin in dog plasma by HPLC with fluorimetric detection

A high throughput, selective and sensitive high-performance liquid chromatographic (HPLC) method for the determination of a water-soluble polymer-bound Camptothecin conjugate (MAG-CPT) and Camptothecin (CPT) in dog plasma has been developed and validated. The method involved the analysis of free and total CPT (free+polymer-bound). Free CPT (intact lactone plus carboxylate) was extracted from acidified plasma using Oasis™ SPE material in 96-well plates. For the assay of the total CPT, plasma proteins were first precipitated with methanol in a 96-well plate containing a 10-μm melt blown polypropylene membrane. The methanolic supernatant was separated and collected into a second 96-well plate by simply applying vacuum to the plate. After hydrolysis at pH 9.8 for 18 h and re-acidification, samples were injected directly from the collection plate onto the HPLC system. MAG-CPT concentration was then calculated by subtraction of free from total CPT. The LLOQs of the method were 1.17 ng/ml for free CPT and 103.10 ng/ml (as CPT equivalent) for MAG-CPT using 0.1 and 0.05 ml of plasma, respectively. Linearity, precision, accuracy and recovery of the method were evaluated. The stability of MAG-CPT in plasma alone and after its stabilisation was carefully evaluated. No interference from blank dog, mouse and human plasma was observed. The suitability of the method for in vivo samples was assessed by the analysis of samples obtained from dogs that had received a single and 5-day repeated dose of MAG-CPT.

A radioreceptor assay for the analysis of AT 1-receptor antagonists : Correlation with complementary LC data reveals a potential contribution of active metabolites

A reliable and sensitive radioreceptor assay based on rat lung homogenate as receptor preparation was developed to determine the angiotensin-II antagonistic profile of losartan and its main active metabolite EXP 3174 as well as its congeners exemplified by UP 269-6 and SL 91.0102-90 DL. This method proved to be precise with an intra- and interday variability of less than 10% and a limit of quantification ≤1 ng ml −1. The analysis of the K i values in protein-free Hepes-buffer versus blank human or rat plasma revealed the distinct high plasma–protein binding of EXP 3174 which consequently caused a dramatic drop of potency from 10–15-fold in the buffer to only about 2-fold in control plasma, when compared to the parent compound losartan and the two congeners investigated. Upon evaluation of clinical samples by both the reported radioreceptor assay (RRA) and the established high-performance liquid chromatography (HPLC), the correlation of the normalized data pairs (concentration equivalents) suggested the contribution of active metabolites to the angiotensin-II antagonistic effect of SL 91.0102-90 DL, but not to the effect of UP 269-6. In the context of an extended preclinical study in rats, the correlation of RRA with the respective HPLC concentration equivalents of losartan and its main active metabolite EXP 3174 confirmed previous findings that only losartan and EXP 3174 exert the angiotensin-II-AT 1 receptor blockade without the contribution of other metabolites (P.C. Wong, W.A. Price, A.T. Chiu et al., J. Pharmacol. Exp. Ther. 255 (1990) 211–217).

Determination of PNU 153429, a new polysulphonated derivative of distamycin A, in rat plasma by reversed-phase ion-pair high-performance liquid chromatography with ultraviolet detection

A rapid and selective ion-pair high-performance liquid chromatographic (HPLC) method for the determination of 2,2′-(carbonylbis(imino- N-methyl-4,2-pyrrolecarbonylimino( N-methyl-4,2-pyrrole)carbonylimino))-bis(1,5-naphtalenedisulphonic acid), tetrasodium salt (PNU 153429, I) in rat plasma has been developed. I is a new drug currently under investigation for the treatment of rheumatoid arthritis. Aliquots of 100 μl of plasma spiked with 10 μl of internal standard solution (PNU 145156E, I.S.) were added to 100 μl of acetonitrile and vortex mixed. After centrifugation, diluted aliquots of the supernatant were transferred to autosampler vials and analyzed by reversed-phase ion-pair liquid chromatography under isocratic conditions. The retention times of I.S. and I were ≈8 and 12 min, respectively. Quantitation was achieved by ultraviolet detection at 323 nm. The assay had a limit of quantitation of 0.1 μg ml −1 when 100 μl of plasma were analyzed. The linearity, precision and accuracy of the method were evaluated. No interference from blank rat, mouse, dog, monkey and human plasma was observed. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from three cannulated male rats that had received a single 100 mg kg −1 i.v. dose of the test compound.

Determination of turosteride, a new inhibitor of 5α-reductase, in human plasma by high-performance liquid chromatography with ultraviolet detection

A sensitive and specific HPLC method for the determination of turosteride in human plasma was developed and validated. Turosteride was extracted from plasma with diethyl ether. Further purifications of the fraction extracted were performed sequentially by solid-phase extraction using a CN cartridge and by liquid—liquid partition between n-hexane and acetonitrile. Finally the acetonitrile solution containing the test compound was evaporated to dryness and the residue dissolved in the mobile phase, then injected onto the HPLC system. The chromatographic separation was performed isocratically by a reversed-phase column filled with ODS using a water—acetonitrile—methanol mixture. The eluate was monitored at 210 nm. No peak interfering with that of turosteride was observed when blank human plasma was assayed. Linearity was obtained in the turosteride concentration range of 5–1000 ng/ml plasma. Six calibration curves in plasma prepared and run on six different days showed correlation coefficients higher than 0.99 and good reproducibility of the slope (C.V.=4.3%). The intra-day precision, evaluated at three concentrations (in the low, mid and high range of the standard curve) and expressed as C.V., ranged from 0.81 to 13.25%. The inter-day precision evaluated at the same concentrations was better than 10.7%. The inter-day accuracy evaluated in the same samples and expressed as the ratio of found/added amount of turosteride ranged from 97.66 to 98.38%. The limit of quantitation was 5 ng/ml plasma. The HPLC method described was successfully employed for the determination of turosteride in plasma samples obtained during a phase I clinical trial with the test compound.

Quantitation of N,N-dimethyltryptamine and harmala alkaloids in human plasma after oral dosing with ayahuasca.

Harmine, harmaline, tetrahydroharmine (THH), and N,N-dimethyltryptamine (DMT) were quantitated in plasma from 15 healthy male volunteers after the ingestion of ayahuasca, a beverage that has been used for religious purposes in Brazil since pre-Columbian times. A growing awareness of the interest in this ancient shamanistic practice in modern urban cultures and the widespread popular dissemination of the inebriant effects and type and sources of the plant admixtures used to prepare the beverage have provided additional impetus for this study. The three harmala alkaloids were quantitated from protein-precipitated plasma by high-performance liquid chromatography using fluorescence detection. Recovery from blank human plasma was quantitative, and the limit of quantitation (LOQ) was below 2 ng/mL of plasma for each of the harmala alkaloids. Standard concentrations ranged from 10 to 250 ng/mL for harmine and THH and from 1.0 to 25.0 ng/mL for harmaline, respectively. Linearity was observed for harmine, harmaline, and THH within these respective ranges. The highest concentrations of harmala alkaloids in human plasma were found to be 222.3 ng/mL for harmine, 134.5 ng/mL for THH, and 9.4 ng/mL for harmaline. DMT was quantitated by gas chromatography using nitrogen-phosphorus detection after liquid-liquid extraction with diphenhydramine as an internal standard. DMT recovery was quantitative, and the limit of detection and LOQ were 0.5 and 5 ng/mL, respectively. Linearity for DMT was observed from 5 to 1000 ng/mL. The one-step extraction method for DMT and the protein precipitation method for the three harmala alkaloids afford rapid, sensitive, and quantitative analyses of these alkaloids with minimal analyte loss. The analytical methods also may be applicable to other matrices, including whole blood and urine samples and homogenized tissue specimens. These are the first reported observations of DMT and harmala alkaloids in plasma after ritual ingestion of ayahuasca.