Quantitative liquid chromatographic and tandem mass spectrometric determination of vitamin D3 in human serum with derivatization: a comparison of in-tube LLE, 96-well plate LLE and in-tip SPME.

Sensitive and selective methods based on high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection were developed for the determination of vitamin D(3) in human serum. Derivatization of vitamin D(3) and its stable isotope labeled internal standard provided highly sensitive quantification and selective detection from endogenous compounds. Samples were prepared using the in-tube liquid-liquid extraction (LLE), 96-well plate LLE, and in-tip solid phase micro-extraction (SPME) in 96-well format. In all methods, the MS/MS detection was performed using Applied Biosystems-Sciex API 3000 tandem mass spectrometers interfaced with a heated nebulizer probe and operated in the positive ionization mode. Both tube and plate LLE methods achieved a lower limit of quantitation (LLOQ) of 0.5 ng/mL when 1.0 and 0.4 mL of human serum was processed, respectively, and were validated in the concentration range of 0.5-25 ng/mL; while for the in-tip SPME method, LLOQ was 5 ng/mL with only 0.1 mL of human serum required. Comparisons were made among three different methods, including precision and accuracy, sample throughput, recovery and matrix effects.

Determination of dextromethorphan and its metabolite dextrorphan in human urine using high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry: a study of selectivity of a tandem mass spectrometric assay.

Analytical method for the simultaneous determination of dextromethorphan (1) and dextrorphan (2) in urine, based on solid-phase extraction of drug from acidified hydrolyzed biological matrix, were developed. The analytes (1 and 2) and the internal standard (levallorphan, 3, IS) were detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) in positive ionization mode using a heated nebulizer (HN) probe and monitoring their precursor-->product ion combinations of m/z 272-->215, 258-->201, and 284-->201 for 1, 2, and 3, respectively, in multiple reaction monitoring mode. The analytes and IS were chromatographed on a Keystone Prism reverse phase (50 mm x 2.0 mm) 5 microm column using a mobile phases consisting of a 35/65 or 27/73 mixtures of methanol/water containing 0.1% TFA adjusted to pH 3 with ammonium hydroxide pumped at 0.4 ml/min for 1 and 2, respectively. The limits of reliable quantification of 1 and 2 were 2 and 250 ng/ml, respectively, when 1 ml of urine was processed. The absence of matrix effect was demonstrated by analysis of neat standards and standards spiked into urine extracts originating from five different sources. The linear ranges of the assay were 2-200 and 250-20,000 ng/ml for 1 and 2, respectively. Assay selectivity was evaluated by monitoring the "cross-talk" effects from other metabolites into the MS/MS channels used for monitoring 1, 2, and 3. In addition, an interfering peak originating from an unknown metabolite of 1 into the quantification of dextromethorphan was detected, requiring an effective chromatographic separation of analytes from other metabolites of 1. The need for careful assessment of selectivity of the HPLC-MS/MS assay in the presence of metabolites, and the assessment of matrix effect, are emphasized.

Simultaneous determination of Aprepitant and two metabolites in human plasma by high-performance liquid chromatography with tandem mass spectrometric detection.

A method for the simultaneous determination of Aprepitant, I (5-[[2(R)-[1(R)-(3,5-bistrifluoromethylphenyl)ethoxy]-3(S)-(4-fluorophenyl) morpholin-4-yl]methyl]-2,4-dihydro-[1,2,4]triazol-3-one) and two active metabolites (II and III) in human plasma has been developed. The method was based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in positive ionization mode using a heated nebulizer interface. The analytes and internal standard (IV) (Fig. 1) were isolated from basified plasma using liquid-liquid extraction. The organic extracts were dried, reconstituted in mobile phase and injected into the HPLC-MS/MS system. The analytes were chromatographed on a narrow bore (50 mm x 2.0 mm, 3 microm) Keystone Scientific's Prism R.P. analytical column, with mobile phase consisting of acetonitrile (ACN):water containing trifluoroacetic acid with pH adjusted to 3 (40:60, v/v) pumped at a flow rate of 0.5 ml/min. The MS-MS detection was performed on a Sciex API 3000 tandem mass spectrometer operated in selected reaction monitoring mode. The precursor-->product ion combinations of m/z 535-->277, 438-->180, 452-->223 and 503-->259 were used to quantify I, II, III, and IV, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10-5000 ng/ml for I and II and 25-5000 ng/ml for III when 1 ml of plasma was processed. The precision of the assay (expressed as coefficient of variation, CV) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. Matrix effect experiments were performed to demonstrate the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. This assay was utilized to support a clinical study where multiple oral doses of I were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of Aprepitant. Concentrations of the two most active metabolites, which if present in high concentrations would increase the neurokinin-1 (NK1) receptor occupancy level and therefore potentially contribute to the antiemetic action of Aprepitant, were determined.

Determination of a novel substance P inhibitor in human plasma by high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometric detection using single and triple quadrupole detectors.

Methods based on high-performance liquid chromatography (HPLC) with atmospheric-pressure chemical ionization (APCI) mass spectrometric (MS) detection using either single (MS) or triple (MS/MS) quadrupole mass spectrometric detection for the determination of (2R)-[1(R)-(3,5-bis-trifluoromethylphenyl)ethoxy]-3(S)-(4-fluoro-phenyl)morpholin-4-ylmethyl]-5-oxo-4,5-dihydro-[1,2,4]triazol)methyl morpholine (Aprepitant, Fig. 1) in human plasma has been developed. Aprepitant (I) and internal standard (II, Fig. 1) were isolated from the plasma matrix buffered to pH 9.8 using a liquid-liquid extraction with methyl-t-butyl ether (MTBE). The analytes were separated on a Keystone Scientific's Javelin BDS C-8 2 mm x 4.6 mm 3 microm guard column coupled to BDS C-8 50 mm x 4.6 mm 3 microm analytical column, utilizing a mobile phase of 50% acetonitrile and 50% water containing 0.1% formic acid and 10 mM ammonium acetate delivered at a flow rate of 1 ml/min. The single quadrupole instrument was operated in a single ion monitoring (SIM) mode analyzing the protonated molecules of Aprepitant and II at m/z 535 and 503, respectively. The triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode (MRM) monitoring the precursor --> ion combinations of m/z 535 --> 277 and 503 --> 259 for Aprepitant and II, respectively. The linear calibration range for both single and triple quadrupole detectors was from 10 to 5000 ng/ml of plasma with coefficients of variation less than 8% at all concentrations. Both single and triple quadrupole instruments yielded similar precision and accuracy results. Matrix effect experiments performed on both instruments demonstrated the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. Both instruments were used successfully to support numerous clinical trials of Aprepitant.

Determination of a cyclooxygenase II inhibitor in human plasma by capillary gas chromatography with mass spectrometric detection.

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of a cyclooxygenase II (COX-II) inhibitor (3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one, I) in human plasma, in two concentration ranges of 0.1-20 and 5-1000 ng/ml, are described. Following liquid-liquid extraction, the residue, after evaporation of the organic phase to dryness, was reconstituted in acetonitrile (20 l) and part of the extract (1 l) was analyzed by GC/MS/SIM. The drug (I) and internal standard (II) were separated on a 25 mx0.2 mm capillary column with HP Ultra 1 (100% dimethylpolysiloxane, 0.33 m) phase and analyzed by MS/SIM monitoring ions at m/z 237 and 282 for I and II, respectively. The standard curve was linear within the lower concentration range of 0.1-20 ng/ml and the lower limit of quantification (LLOQ) in plasma was 0.1 ng/ml. Intraday coefficients of variation (CV, n=5) were 8.9, 4.2, 5.7, 3.1, 1.9, 1.9, and 4.4% at 0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 ng/ml, respectively. The standard curve was also linear within the higher concentration range of 5-1000 ng/ml and the LLOQ in plasma was 5 ng/ml. Intraday coefficients of variation (CV, n=5) were all below 9% at all concentrations within the standard curve range. The accuracy for I in human plasma was 91-112% and the recovery of I and II was greater than 70% at all concentrations within both standard curve ranges. The details of the assay methodology are presented.

Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS.

In recent years, high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection has been demonstrated to be a powerful technique for the quantitative determination of drugs and metabolites in biological fluids. However, the common and early perception that utilization of HPLC-MS/MS practically guarantees selectivity is being challenged by a number of reported examples of lack of selectivity due to ion suppression or enhancement caused by the sample matrix and interferences from metabolites. In light of these serious method liabilities, questions about how to develop and validate reliable HPLC-MS/MS methods, especially for supporting long-term human pharmacokinetic studies, are being raised. The central issue is what experiments, in addition to the validation data usually provided for the conventional bioanalytical methods, need to be conducted to confirm HPLC-MS/MS assay selectivity and reliability. The current regulatory requirements include the need for the assessment and elimination of the matrix effect in the bioanalytical methods, but the experimental procedures necessary to assess the matrix effect are not detailed. Practical, experimental approaches for studying, identifying, and eliminating the effect of matrix on the results of quantitative analyses by HPLC-MS/MS are described in this paper. Using as an example a set of validation experiments performed for one of our investigational new drug candidates, the concepts of the quantitative assessment of the "absolute" versus "relative" matrix effect are introduced. In addition, experiments for the determination of, the "true" recovery of analytes using HPLC-MS/MS are described eliminating the uncertainty about the effect of matrix on the determination of this commonly measured method parameter. Determination of the matrix effect allows the assessment of the reliability and selectivity of an existing HPLC-MS/MS method. If the results of these studies are not satisfactory, the parameters determined may provide a guide to what changes in the method need to be made to improve assay selectivity. In addition, a direct comparison of the extent of the matrix effect using two different interfaces (a heated nebulizer, HN, and ion spray, ISP) under otherwise the same sample preparation and chromatographic conditions was made. It was demonstrated that, for the investigational drug under study, the matrix effect was clearly observed when ISP interface was utilized but it was absent when the HN interface was employed.

Determination of efavirenz, a selective non-nucleoside reverse transcriptase inhibitor, in human plasma using HPLC with post-column photochemical derivatization and fluorescence detection.

Methods for the quantitative determination of efavirenz in human plasma and the qualitative assessment of the stereochemical integrity of efavirenz in post-dose human plasma samples are described. After the addition of an internal standard, plasma samples were extracted with hexane-methylene chloride (65/35, v/v%). The extracts were evaporated to dryness and reconstituted in mobile phase. Upon exposure to UV light, the analyte was found to form fluorescent products; the major fluorescent product was isolated and identified as a substituted quinoline. Thus, the plasma extracts were analyzed via HPLC with post-column photochemical derivatization and fluorescence detection. Reverse phase chromatography was used for the quantitative assay, whereas chromatography with a column containing a chiral stationary phase (dinitrobenzoyl leucine) was used for the stereochemical assessment. The quantitative assay has been validated in the concentration range of 50-1000 ng/ml using 0.5 ml samples. Analyte recovery was better than 89% at all points on the standard curve. Intra-day precision was better than 5% C.V., while accuracy was between 95 and 104% of nominal over the range of the assay. The selective detection method reduces the likelihood of interference by co-administered medications or endogenous species. The stereochemical configuration of efavirenz was confirmed to remain intact in post-dose human plasma samples. The quantitative method has been successfully utilized to support a study in which a possible drug interaction between co-administered HIV protease inhibitors and efavirenz was evaluated.

High-performance liquid chromatographic-tandem mass spectrometric evaluation and determination of stable isotope labeled analogs of rofecoxib in human plasma samples from oral bioavailability studies.

A method for the simultaneous determination of a cyclooxygenase-2 inhibitor, 4-(4-methanesulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib, I) and [13C7]rofecoxib, (II), in human plasma has been developed to support the clinical oral bioavailability (BA) study of I. The method is based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in the negative ionization mode using a heated nebulizer interface. Two different stable isotope labeled analogs of I were initially evaluated for their use as intravenous (i.v.) markers in the BA study. [13CD3]Rofecoxib was shown to be isotopically unstable in plasma and water containing solvent and an efficient deuterium exchange prevented its use in the study. On the other hand, the isotopic integrity of the subsequently synthesized [13C7]rofecoxib (II) was maintained, as expected, in plasma and other solvent systems. The results of these experiments clearly demonstrated the need for the careful evaluation of the isotopic integrity of the stable isotope labeled compound for the successful utilization of these compounds in BA studies and also as internal standards in the quantitative analysis of drugs in biological fluids. After liquid-liquid extraction of I, II, and internal standard (III) from plasma, the analytes were chromatographed on a narrow bore (100 mm x 3.0 mm) C18 analytical column, with mobile phase consisting of acetonitrile-water (1:1, v/v) at a flow-rate of 0.5 ml/min. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in the selected reaction monitoring mode. The precursor-->product ion combinations of m/z 313-->257, 320-->292, and 327-->271 were used to quantify I, II, and III, respectively. The assay was validated in the concentration range of 0.1 to 100 ng/ml of plasma for both I and II. The precision of the assay (expressed as relative standard deviation) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. The assay was utilized to support the clinical BA study in which oral doses of I were administered together with an i.v. dose of II to determine the oral BA of rofecoxib at 12.5- and 25-mg doses.

Determination of rofecoxib (MK-0966), a cyclooxygenase-2 inhibitor, in human plasma by high-performance liquid chromatography with tandem mass spectrometric detection.

A method for the determination of 4-(4-methanesulfonylphenyl)-3-phenyl-5H-furan-2-one (Rofecoxib, Vioxx, MK-0966, I) a cyclooxygenase-2 inhibitor, in human plasma has been developed. The method is based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in negative ionization mode using a heated nebulizer interface. Drug and internal standard (II) were isolated from basified plasma using liquid-liquid extraction. The organic extracts were dried, reconstituted in mobile phase and injected into the HPLC-MS-MS system. Compounds I and II were chromatographed on a narrow bore (100 mm x 3.0 mm) C18 analytical column, with mobile phase consisting of acetonitrile:water (1:1, v/v) at a flow-rate of 0.4 ml/min. The MS-MS detection was performed on a PE-Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode. The parent-->product ion combinations of m/z 313-->257 and 327-->271 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 0.1 to 100 ng/ml of plasma. The precision of the assay (expressed as coefficient of variation) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. The effect of HPLC mobile phase components on the ionization efficiency and sensitivity of detection in the positive and negative ionization modes, and the detailed description of all necessary steps involved in the assay for I in plasma are presented.

Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion spray and turbo ion spray tandem mass spectrometric detection.

Methods for the determination of a semi-synthetic cyclic hexapeptide (I, MK-0991) in human plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection using pneumatically assisted electrospray (ion spray, ISP) and turbo ion spray (TISP) interfaces were developed. Drug and internal standard (II, an isostere of I) were isolated from plasma by solid-phase extraction (SPE). The eluent from SPE was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The use of ISP, TISP and heated nebulizer (HN) interfaces as sample introduction systems were evaluated and showed that the heated nebulizer was not adequate for analysis due to thermal instability and/or adsorption of I and II to glass surfaces of the interface. Compounds I and II were chromatographed on a wide pore (300 A), 150x4.6 mm C8 analytical column, and the HPLC flow-rate of 1.2 ml/min was split 1:20 prior to introduction to the ISP or TISP interface of the mass spectrometric system. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode (SRM). The precursor-->product ion combinations of m/z 1093.7-->1033.6 and 1094.7-->1033.6 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10-1000 ng/ml using ISP, and 2.5-500 ng/ml of plasma using TISP with good precision and adequate accuracy. The effects of HPLC mobile-phase components on the ionization efficiency and sensitivity of detection in the positive ionization mode, the evaluation of the matrix effect, and limitations in sensitivity of detection of I due to the formation of multiply charged species are presented.

Development of high-performance liquid chromatography-tandem mass spectrometric methods for the determination of a new oxytocin receptor antagonist (L-368,899) extracted from human plasma and urine: a case of lack of specificity due to the presence of metabolites.

The purpose of this work was to develop HPLC-MS-MS methods for the quantification of L-368,899 (1) in human plasma and urine and to evaluate the selectivity of these methods in post-dose samples in the presence of metabolites. Assays were based on double liquid-liquid extraction of the drug and internal standard (I.S., 2) from basified plasma, evaporation of the extracts to dryness, derivatization of the primary amino groups of 1 and 2 with trifluoroacetic anhydride (TFAA) to form trifluoroacetylated (TFA) analogs, and HPLC analysis using tandem mass spectrometer equipped with the heated nebulizer interface as a detector. The derivatization with TFAA was required to eliminate the carryover and adsorption problems encountered when underivatized molecules were chromatographed, and allowed quantitation at low concentration (0.5 ng/ml) in plasma and urine. Initially, assays in control human plasma and urine were validated in the concentration range of 0.5-75 ng/ml, using simplified chromatographic conditions with a 2-min run-time and no separation of the drug from I.S.. Quantitation was based on the high selectivity of detection and multiple reaction monitoring (MRM) using the precursor-->product ion combinations of m/z 651-->152 and m/z 665-->425 for the TFA-derivatized 1 and 2, respectively. However, when selected post-dose urine samples from a clinical study were analyzed using this assay, the area of the I.S. peak was 4 to 7 times larger than the area of I.S. peak in pre-dose urines, indicating the presence of metabolites giving rise to the m/z 665-->425 I.S. peak. A number of metabolites contributing to the I.S. ion pair were separated from 1 and 2 using a longer analytical column, a weaker mobile phase, and by extending the HPLC run-time to 12 min. Under these new conditions, the modified assays both in plasma and urine were validated in the concentration range of 0.5 to 75.0 ng/ml. These assays were selective in the post-dose urine samples in the presence of metabolites.

Determination of a novel selective inhibitor of type 1 5alpha-reductase in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry.

A sensitive and specific assay of human plasma for the determination of (5alpha,7beta,16beta)-16[(4-chlorophenyl)oxy]-4,7- dimethyl-4-aza-androstan-3-one (I), a selective inhibitor of human type 1 5alpha-reductase, has been developed. The method is based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. The analyte (I) and internal standard, Proscar (II), were isolated from the basified biological matrix using a liquid-liquid extraction with methyl-tert.-butyl ether (MTBE). The organic extract was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring using the precursor-->product ion combinations of m/z 430-->114 and 373-->305 was used to quantify I and internal standard (II), respectively. The assay was validated in the concentration range of 0.5 to 500 ng/ml in human plasma. The precision of the assay, expressed as coefficient of variation (C.V.), was less than 7% over the entire concentration range, with adequate assay specificity and accuracy. The HPLC-MS-MS method provided sufficient sensitivity to completely map the 24 h pharmacokinetic time-course following a single 0.5 mg dose of I.

Matrix effect in quantitative LC/MS/MS analyses of biological fluids: a method for determination of finasteride in human plasma at picogram per milliliter concentrations.

Contrary to common perceptions, the reliability of quantitative assays for the determination of drugs in biological fluids using high-performance liquid chromatography with tandem mass spectrometric (LC/MS/MS) detection methods and the integrity of resulting pharmacokinetic data may not be absolute. Results may be adversely affected by lack of specificity and selectivity due to ion suppression caused by the sample matrix, interferences from metabolites, and "cross-talk" effects. In this paper, an example of the effect of the sample matrix on the determination of finasteride (I) in human plasma is presented. The ion suppression effect was studied by analyzing standards of I injected directly in mobile phase and comparing the response (peak areas) of I and an internal standard (II) with the peak areas of the same analytes spiked before extraction into five different plasma pools and standards spiked into the plasma extracts after extraction. The LC/MS/MS analyses were performed using a turbo ion spray interface (TISP) under chromatographic conditions, characterized by minimal (total run time of 2 min, capacity factors, k' of 1.50 and 1.75 for I and II, respectively) and high retention of the analytes (total run time 6 min, k' of 3.25 and 13.25 for I and II, respectively). The absolute peak areas for I and II in different plasmas were calculated, and the slopes and peak area ratios at all concentrations within the standard curve ranges were compared. When analyses were performed under conditions of minimal HPLC retention, the slope of the standard line for one set of plasma samples was substantially different (about 50% higher) from that from other plasma sources. The precision of the assay, expressed as coefficient of variation (CV, %) was also inadequate and varied from 15 to 30% at all concentrations within the standard curve range. When the same experiments were repeated using high HPLC retention, the slopes from different plasma sources were practically the same, and the CV was improved to 6-14%. By increasing k' and providing more chromatographic retention of analytes, the "unseen" interferences from plasma matrix were mostly separated from analytes, practically eliminating the ion suppression. In addition, by eliminating from plasma extracts a number of endogenous components through more selective extraction, the ion suppression was also minimized. The detailed data and the design of these experiments are presented. In addition, development of a highly sensitive assay for I in human plasma at low picogram per milliliter concentrations using LC/MS/MS with a heated nebulizer (HN) interface, instead of a TISP interface, is described. In this case, the effects of sample matrixes were not observed.

Determination of a substance P antagonist in human plasma and urine using high-performance liquid chromatography with ultraviolet absorbance and tandem mass spectrometric detection.

A high-performance liquid chromatographic (HPLC) assay using ultraviolet (UV) detection was developed and compared with a HPLC method with tandem mass spectrometric (HPLC/MS-MS) detection for the determination of a substance P receptor antagonist 2(S)-((3,5-bis(trifluoromethyl)benzyl)-oxy)-3(S)-phenyl-4-((3-oxo-1,2,4- triazol-5-yl) methyl)morpholine (Fig. 1, Ia, L-742 694) in human plasma and urine. The drug was isolated from the biological matrix through liquid-liquid extraction. In the HPLC/UV method, the samples were initially injected onto a cyano Hypersil column, and the chromatographic region containing the peaks of interest was heart-cut onto an analytical C-18 Hypersil column via a column switching device. The analyte was quantified by monitoring absorbance at 205 nm. The limit of quantification for I extracted from 1 ml of plasma or urine was 2.5 ng ml-1, and the assays were validated in the concentration range 2.5-500 ng ml-1. The HPLC/MS-MS method were validated in the concentration range 0.2-500 ng ml-1. Both assays provided data with precision, measured as coefficient of variation, better than 10% at all points within the standard curve range and with adequate accuracy.

Low level determination of a novel 4-azasteroid and its carboxylic acid metabolite in human plasma and semen using high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry.

Compound I (4.7beta-dimethyl-4-azacholestan-3-one, MK-0386) is a potent 5alpha-reductase type 1 (5alphaR1) inhibitor. Sensitive (0.2 ng/ml), specific and separate assays have been developed and validated for the analysis of I and its carboxylic acid metabolite (II) in human semen and plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. After liquid-liquid extraction of the analytes from biological matrix, the extracts were chromatographed on a short (50 mm) analytical column during analysis of I, and on a longer (150 mm) column with a weaker mobile phase during the analysis of II. This additional chromatographic separation was required to separate II from a secondary metabolite present in post-dose plasma samples interfering with the quantification of II. The MS-MS detection was performed on a Sciex API III Plus tandem mass spectrometer using the heated nebulizer probe. Monitoring the parent-->product ion combinations of m/z 416-->114 and 404-->114, in the multiple reaction monitoring (MRM) mode, after chromatographic separation, allowed quantification of both analytes. The standard curve in plasma was linear in the concentration range of 0.2 to 200 ng/ml for both I and II with correlation coefficients greater than 0.99 and coefficients of variation of less than 15% for replicate (n=5) analysis at all concentrations within the standard curve range. For the semen assay the linear range for determination of I was from 0.2 to 50 ng/ml. These assays were applied to support a number of clinical studies with I and their validity and long-term performance was confirmed during analyses of clinical samples from these studies. The need for careful assessment of the specificity of MS-MS assays in post-dose biological fluid samples in the presence of metabolites was emphasized.

Determination of a novel growth hormone secretagogue (MK-677) in human plasma at picogram levels by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry.

A sensitive and specific method for the determination of N-[1(R)¿[1,2-dihydro-1-methylsulfonylspiro(3H-indole-3,4'-piper idin)-1'-yl]carbonyl¿-2-(phenylmethoxy)ethyl]-2-amino-2-meth ylpropanamide (MK-677, I), a growth hormone secretagogue, has been developed. The method is based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. The analyte and internal standard (II) were isolated from the basified plasma using a liquid-liquid extraction with methyl-tert.-butyl ether (MTBE). The organic extract was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring of parent-->product ion combinations at m/z 529-->267 and 527-->267 was used to quantify I and II, respectively. The assay was validated in human plasma in the concentration range of 0.1 to 100 ng/ml, and the limit of quantification (LOQ) was 0.1 ng/ml. The precision of the assay, as expressed as coefficients of variation (C.V.,%) was less than 7% at all concentrations within the standard curve range, with adequate assay specificity and accuracy. The HPLC-MS-MS method provided sufficient sensitivity to completely map the pharmacokinetic time-course following a single 5-mg oral dose of I.

Low level determination of dorzolamide and its de-ethylated metabolite in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry.

A sensitive and specific method for the determination of dorzolamide (I) and its de-ethylated metabolite (II) in human plasma has been developed utilizing high pressure liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection. The analytes and internal standard (III) were isolated from the deproteinized pH 8.0 buffered plasma, using a liquid-liquid extraction with a mixture of ethyl acetate, toluene, and isopropanol. The analytes were then back extracted into 0.085% phosphoric acid (200 microliters) and after washing the acidic extract with hexane, the organic layer was discarded and a fraction (50 microliters) of the acid extract was injected into the LC/MS/MS system. The MS/MS detection was performed on a PE Sciex API III tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring of the parent-->product ion combinations of m/z 325-->199, 297-->199, and 397-->306 were used to quantify I, II, and III, respectively. The assay was validated in the concentration ranges of 0.5-100 and 2.5-100 ng ml-1 of plasma for I and II, respectively. The precision of the assays, expressed as coefficients of variation (C.V.%), were less than 10% over the entire concentration range, with adequate assay specificity and accuracy. The LC/MS/MS method provided a 10-fold increase in the sensitivity of I over the previously reported HPLC/UV method [1].

Picogram determination of a novel dopamine D4 receptor antagonist in human plasma and urine by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry.

A sensitive and specific assay for the determination of 3-[[4-(4-chlorophenyl)piperazin-1-yl]methyl]-1H-pyrrolo[2,3-b]p yri dine (I, L-745,870), a potential antipsychotic agent, has been developed, utilizing high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. The analyte and the internal standard (II, 3-[[4-(4-trifluoromethyl)piperazin-1-yl]methyl]-1H-pyrrolo[2,3-b] pyridine) were isolated from a basified biological matrix using liquid-liquid extraction with methyl-tert.-butyl ether. The organic extract was evaporated to dryness, the residue was reconstituted in a mobile phase and injected into the HPLC system. The chromatographic conditions used for the analysis were a Keystone Scientific C18 BDS 150 x 4.6 mm, 5 microns column with a mobile phase consisting of a 40:60 (v/v) mixture of acetonitrile and water containing 10 mM ammonium acetate and 0.1% formic acid pumped at a flow-rate of 1.2 ml/min, yielding retention times of 3.4 and 5.0 min for I and II, respectively. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring using the parent-->product ion combinations of m/z 327-->131 and 361-->131 were utilized to quantitate I and II, respectively. The assays were validated in the concentration range of 0.1 to 100 and 0.5 to 500 ng/ml for plasma and urine, respectively. The precision of the assays, expressed as coefficients of variation were less than 10% over the entire concentration range, with adequate assay accuracy, sensitivity and specificity to determine the pharmacokinetics in human subjects following a single 1-mg dose.

Effect of MK-386, a novel inhibitor of type 1 5 alpha-reductase, alone and in combination with finasteride, on serum dihydrotestosterone concentrations in men.

Two isozymes (types 1 and 2) of 5 alpha-reductase (5 alpha R; EC 1.3.99.5), with differential tissue distribution, have been identified in humans. These enzymes catalyze the reduction of testosterone (T) to dihydrotestosterone (DHT). The contributions of each of these isozymes to serum and tissue concentrations of DHT remain to be fully defined. Finasteride, a selective inhibitor of type 2 5 alpha R, lowers circulating DHT levels by approximately 70% in men after treatment with 5 mg daily. MK-386 (4,7 beta-dimethyl-4-aza-5 alpha-cholestan-3-one) is a new selective inhibitor of type 1 5 alpha R. A single rising dose, alternating panel, trial in 16 healthy males (age range, 21-25 yr) studied the effect of 0.1-100 mg MK-386. DHT was maximally reduced by 20-30% relative to placebo at MK-386 doses of 10 mg or more, orally, by 24 h posttreatment (P < 0.01 vs. placebo). No consistent effect on T concentrations was evident. In a second trial, finasteride (5 mg) was given for 19 days to 10 healthy young men (age range, 24-47 yr); a 25-mg dose of MK-386 was added for 2 days of combination therapy after at least 10 days of finasteride treatment. Withdrawal of MK-386 was followed by 5-6 days of finasteride follow-up treatment. Finasteride alone reduced DHT, on the average, by 68.7% (SE = 3.4%). Addition of MK-386 suppressed DHT by 89.5% (SE = 1.4%) relative to baseline (P < 0.01 vs. effect of finasteride alone). Small increases in serum T were observed with finasteride alone and in combination with MK-386 (approximately 10% and 19%, respectively). These data are consistent with selective 5 alpha R type 1 inhibition in man by MK-386 and the prediction that types 1 and 2 5 alpha R account for all, or nearly all, of circulating DHT. Further clinical trials are needed to assess the therapeutic utility of type 1 5 alpha R inhibition as well as that of combined inhibition of types 1 and 2 5 alpha R.

Determination of pirenzepine in human plasma using liquid chromatography with tandem mass spectrometric detection.

A sensitive (LOQ = 1 ng ml-1) and specific method based on liquid chromatography with tandem mass spectrometric (MS/MS) detection has been developed and validated for the analysis of pirenzepine (I) in plasma. Sample preparation involved liquid-liquid extraction of drug and internal standard (IS) from basified plasma. The organic extract was evaporated to dryness, and the residue was reconstituted in the mobile phase and then injected into the liquid chromatography/MS/MS system. Drug, IS, and endogenous impurities were separated using reverse-phase chromatography. A Sciex API III tandem mass spectrometer equipped with a heated nebulizer was operated in the positive ion mode. Multiple reaction monitoring using the parent-->daughter ion combinations of m/z 352-->113 and 629-->422 was used to quantify I and IS, respectively. The method was validated in the concentration range of 1-100 ng ml-1 plasma with adequate assay precision and accuracy, and was utilized to support human safety and tolerability study with I.