Semi-automated determination of plasma stability of drug discovery compounds using liquid chromatography-tandem mass spectrometry

A simple procedure for the measurement of stability of drug candidates in plasma was developed to eliminate the traditional labor-intensive and time-consuming sample preparation procedures that are typically used for these studies. The procedure makes use of a thermostatic autosampler as an incubator combined with the direct plasma injection method based on high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS-MS). Untreated human, monkey, mouse and rat plasma containing the test compound was directly injected into a mixed-function column for on-line protein removal and chromatography. The test compound and its biotransformation product were separated via HPLC and monitored using the tandem mass spectrometer. The need for adequate chromatographic separation of the test compound (M) from its carboxylic acid metabolite (M+1) is demonstrated. Plasma samples from four different species at specified incubation temperatures were sequentially assayed in one analytical procedure. The injection-to-injection time was about 6 min. The peak responses of the test compound in individual plasma samples were repeatedly determined every 24 min. The retention times and peak shape of all analytes were found to be consistent throughout the experiments. The stability of the test compound in plasma was found to be a function of animal species, incubation time and temperature. The test compound was rapidly degraded in rat plasma at 37 degrees C, but it could be stabilized by adding sodium thiosulfate.     

Multi-component plasma quantitation of anti-hyperglycemic pharmaceutical compounds using liquid chromatography-tandem mass spectrometry

Type-2 diabetes is a disorder characterized by disrupted insulin production leading to high blood glucose levels. To control this disease, combination therapy is often used. Hypoglycemic agents such as metformin, glipizide, glyburide, repaglinide, rosiglitazone, nateglinide, and pioglitazone are widely prescribed to control blood sugar levels. These drugs provide the basis for the development of a quantitative multianalyte bioanalytical method. As an example, a highly sensitive and selective multi-drug method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. This rapid, automated method consists of protein precipitation of 20 microL of plasma coupled with gradient HPLC elution of compounds using 10 mM ammonium formate buffer and 0.1% formic acid in acetonitrile as the mobile phases. MS/MS detection was performed using turbo ion spray in the positive ion multiple reaction monitoring (SRM) mode. A lower limit of quantitation (LLQ) in a range of 1.0-5.0 ng/mL was achieved for all analytes. The linearity of the method was observed over a 500-fold dynamic range. Drug recoveries ranged from 86.2 to 94.2% for all analytes of interest. Selectivity, sample dilution, intra-day and inter-day accuracy and precision, and stability assessment were evaluated for all compounds.     

Doping control analysis of trenbolone and related compounds using liquid chromatography-tandem mass spectrometry

Trenbolone (17β-hydroxy-estra-4,9,11-trien-3-one) and its derivatives such as 17α-methyltrenbolone represent a class of highly potent anabolic-androgenic steroids, which are prohibited in sports according to the regulation of the World Anti-Doping Agency (WADA). Due to marginal gas chromatographic properties of these compounds but excellent proton affinities resulting from a large and conjugated π-electron system, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the method of choice for the detection of these analytes in sports drug testing. Recent findings of trenbolone and methyltrenbolone in doping control urine samples of elite athletes demonstrated the importance of a sensitive and robust analytical method, which was based on an enzymatic hydrolysis of target compounds, liquid-liquid extraction and subsequent LC-MS/MS measurement. Diagnostic product ions obtained after collision-induced dissociation of protonated molecules were found at m/z 227, 211, 199 and 198, which enabled targeted screening using multiple reaction monitoring. Using 7 model compounds (trenbolone, epitrenbolone, methyltrenbolone, ethyltrenbolone, propyltrenbolone, 17-ketotrenbolone and altrenogest), the established method was validated for specificity, lower limits of detection (0.3-3 ng/mL), recovery (72-105%), intraday and interday precision (≤20%).     

Determination of benidipine in human plasma using liquid chromatography-tandem mass spectrometry

Fumonisins are water soluble mycotoxins produced by the fungus Fusarium verticillioides (formerly F. moniliforme). Fumonisin B(1) (FB(1)) is a diester of propane-1,2,3-tricarboxylic acid and 2-amino-12, 16-dimethyl-3,5,10,14,15-pentahydroxyeicosane, and is the most abundant of the naturally occurring fumonisins. Upon removal of the two tricarballylic acid side chains, the structure is referred to as hydrolyzed FB(1) (HFB(1)). FB(1) and HFB(1) are structurally similar to sphinganine, a sphingoid base. The fumonisins do not absorb UV light or fluoresce; therefore, derivatizing reagents are used for detection when separation is by high performance liquid chromatography (HPLC). The standard derivatizing reagent used for HPLC is ortho-phthalaldehyde (OPA) plus 2-mercaptoethanol (ME) reaction partner, however, the OPA-FB(1) derivative is not stable at room temperature. The objectives of this study were to: (1). determine the effect of temperature on the stability of the OPA-FB(1) derivative and (2). determine which structural characteristics of FB(1) contribute to the instability of the OPA-FB(1) derivative. The results indicate that OPA-FB(1), OPA-FB(3) and OPA-HFB(1) derivatives are unstable at 24 degrees C but that their stability improves significantly at 4 degrees C. The OPA-sphinganine derivative is stable for at least 24h at 24 degrees C. Thus, the instability of the OPA-FB(1) derivative may be attributed to its lack of a hydroxyl group at the carbon 1 position.     

Enantioselective determination of azelnidipine in human plasma using liquid chromatography-tandem mass spectrometry

A sensitive and simple method was developed for determination of the enantiomers of azelnidipine, (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, in human plasma using chiral liquid chromatography with positive ion atmospheric pressure chemical ionization tandem mass spectrometry. Plasma samples spiked with stable isotope-labeled azelnidipine, [(2)H(6)]-azelnidipine, as an internal standard, were processed for analysis using a solid-phase extraction in a 96-well plate format. The azelnidipine enantiomers were separated on a chiral column containing alpha(1)-acid glycoprotein as a chiral selector under isocratic mobile phase conditions. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, monitoring the transitions from m/z 583-->167 for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, and from m/z 589-->167 for [(2)H(6)]-azelnidipine. The standard curve was linear over the studied range (0.05-20 ng/mL), with r(2)>0.997 using weighted (1/x(2)) quadratic regression, and the chromatographic run time was 5.0 min/injection. The intra- and inter-assay precision (coefficient of variation), calculated from the assay data of the quality control samples, was 1.2-8.2% and 2.4-5.8% for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, respectively. The accuracy was 101.2-117.0% for (R)-(-)-azelnidipine and 100.0-107.0% for (S)-(+)-azelnidipine. The overall recoveries for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine were 71.4-79.7% and 71.7-84.2%, respectively. The lower limit of quantification for both enantiomers was 0.05 ng/mL using 1.0 mL of plasma. All the analytes showed acceptable short-term, long-term, auto-sampler and stock solution stability. Furthermore, the method described above was used to separately measure the concentrations of the azelnidipine enantiomers in plasma samples collected from healthy subjects who had received a single oral dose of 16 mg of azelnidipine.     

Highly sensitive determination of saquinavir in biological samples using liquid chromatography-tandem mass spectrometry

A selective and sensitive method for the determination of the HIV protease inhibitor saquinavir in human plasma, saliva, and urine using liquid-liquid extraction and LC-MS-MS has been developed, validated, and applied to samples of a healthy individual. After extraction with ethyl acetate, sample extracts were chromatographed isocratically within 5 min on Kromasil RP-18. The drug was detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source and 2H(5)-saquinavir as internal standard. The limit of quantification was 0.05 ng/mL. The accuracy of the method varied between -1 and +10% (SD within-batch) and the precision ranged from +4 to +10% (SD batch-to-batch). The method is linear at least within 0.05 and 87.6 ng/mL. After a regular oral dose (600 mg) saquinavir concentrations were detectable for 48 h in plasma and were well correlated with saliva concentrations (r(2)=0.9348, mean saliva/plasma ratio 1:15.1). The method is well suited for low saquinavir concentrations in different matrices.     

Highly sensitive quantification of vancomycin in plasma samples using liquid chromatography-tandem mass spectrometry and oral bioavailability in rats

We developed a highly sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS-MS) for a glycopeptide antibacterial drug, vancomycin (VCM), in rat plasma. After precipitating 100 micro l of plasma with 300 micro l of 10% trifluoroacetic acid-methanol (2:1, v/v), the supernatant was diluted with 300 micro l of distilled water and was passed through a filter. LC-MS-MS equipped with electrospray ionization in the positive ion mode used a pair of ions at 725/144 m/z for VCM in the multiple reaction-monitoring mode with a sample injection volume of 20 micro l. The calibration curve had a linear range from 0.01 to 20 micro g/ml when linear least square regression was applied to the concentration versus peak area plot. The drug in the sample was detected within 5 min. Precision, accuracy and limit of quantitation indicated that this method was suitable for the quantitative determination of VCM in rat plasma. Using this method, we defined for the first time that the oral bioavailability of VCM in rats was 0.069%. This method can be applied to basic pharmacokinetic and pharmaceutical studies in rats.     

Direct analysis of un-derivatized asymmetric dimethylarginine (ADMA) and L-arginine from plasma using mixed-mode ion-exchange liquid chromatography-tandem mass spectrometry

A high-throughput analytical method was developed for the measurement of asymmetric dimethylarginine (ADMA) and L-arginine (ARG) from plasma using LC/MS/MS. The sample preparation was simple and only required microfiltration prior to analysis. ADMA and ARG were assayed using mixed-mode ion-exchange chromatography which allowed for the retention of the un-derivatized compounds. The need for chromatographic separation of ADMA from symmetric dimethylarginine (SDMA) was avoided by using an ADMA specific product ion. As a result, the analytical method only required a total run time of 2 min. The method was validated by linearity, with r2>or=0.995 for both compounds, and accuracy, with no more than 7% deviation from the theoretical value. The estimated limit of detection and limit of quantification were suitable for clinical evaluations. The mean values of plasma ADMA and ARG taken from healthy volunteers (n=15) were 0.66+/-0.12 and 87+/-35 microM, respectively; the mean molar ratio of ARG to ADMA was 142+/-81.     

Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.     

Application of column-switching liquid chromatography-tandem mass spectrometry for the determination of pharmaceutical compounds in tissue samples

Information on plasma-tissue distribution which is important for drug development may be obtained by "in silico" prediction tools. To support the validation of computer models, drug concentrations in rat plasma and tissues (brain, liver, kidney, testes, spleen, gut, lung. heart, muscle, skin and fat) had to be determined. In our work, we established analytical assays for a variety of substances including nicardipine, nitrendipine, felodipine and benzodiazepines. Sample preparation had to be simple and method development as well as analytical run time short to allow a high sample throughput and to minimize resources. Column-switching HPLC after homogenization and protein precipitation served as an efficient, easy and rapid sample preparation method, followed by selective MS-MS detection. Optimization of the trapping procedure was performed in order to reduce the influence of endogenous interferences and to obtain good recovery. Chromatographic separation was necessary to increase the selectivity. The use of small analytical column dimensions (2.1 x 10 mm) was investigated to achieve higher sample throughput without compromising the assay quality. Mass spectrometric parameters, such as ionization modes (positive vs. negative) and ion source types (TurbolonSpray vs. APCI) were screened to find suitable conditions for sensitive analysis of the compounds. Matrix suppression effects were taken into consideration. Calibration samples were prepared in plasma only, whereas quality control samples were prepared in both plasma and tissues to save animals and time. Accuracy and precision were in the range of 84.4-119.1% and 1-16.5%, respectively. Limits of quantification were in the range of 0.5-2.5 ng/ml for plasma and 2-10 ng/ml for tissues. Run times as short as 2.2 min could be achieved.     

Informatics platform for global proteomic profiling and biomarker discovery using liquid chromatography-tandem mass spectrometry

We have developed an integrated suite of algorithms, statistical methods, and computer applications to support large-scale LC-MS-based gel-free shotgun profiling of complex protein mixtures using basic experimental procedures. The programs automatically detect and quantify large numbers of peptide peaks in feature-rich ion mass chromatograms, compensate for spurious fluctuations in peptide signal intensities and retention times, and reliably match related peaks across many different datasets. Application of this toolkit markedly facilitates pattern recognition and biomarker discovery in global comparative proteomic studies, simplifying mechanistic investigation of physiological responses and the detection of proteomic signatures of disease.     

Rapid determination of tafenoquine in small volume human plasma samples by high-performance liquid chromatography-tandem mass spectrometry

A method was developed for the determination of tafenoquine (I) in human plasma using high-performance liquid chromatography-tandem mass spectrometry. Prior to analysis, the protein in plasma samples was precipitated with methanol containing [2H3(15N)]tafenoquine (II) to act as an internal standard. The supernatant was injected onto a Genesis-C18 column without any further clean-up. The mass spectrometer was operated in the positive ion mode, employing a heat assisted nebulisation, electrospray interface. Ions were detected in multiple reaction monitoring mode. The assay required 50 microl of plasma and was precise and accurate within the range 2 to 500 ng/ml. The average within-run and between-run relative standard deviations were < 7% at 2 ng/ml and greater concentrations. The average accuracy of validation standards was generally within +/- 4% of the nominal concentration. There was no evidence of instability of I in human plasma following three complete freeze-thaw cycles and samples can safely be stored for at least 8 months at approximately -70 degrees C. The method was very robust and has been successfully applied to the analysis of clinical samples from patients and healthy volunteers dosed with I.     

Quantitation of five nevirapine oxidative metabolites in human plasma using liquid chromatography-tandem mass spectrometry

A multiple-reaction-monitoring LC/MS/MS method for the analysis of nevirapine oxidative metabolites, 2-hydroxynevirapine, 3-hydroxynevirapine, 8-hydroxynevirapine, 12-hydroxynevirapine, and 4-carboxynevirapine, in human plasma was developed and validated. The metabolites were isolated from 50 microL heparinized plasma by enzymatic hydrolysis of the glucuronide conjugates to the free metabolite followed by protein precipitation with acetonitrile. Peaks were quantitated at 3.03 min for the 4-carboxynevirapine metabolite, at 3.72, 4.27, 5.27, and 5.73 min for the positional 2-hydroxynevirapine, 12-hydroxynevirapine, 3-hydroxynevirapine, and 8-hydroxynevirapine metabolites, respectively, and 2.30 min for the internal standard, pirenzepine. The assay was accurate and precise based on assay validation controls over the nominal range of 0.010-1.0 mg/L. The average accuracy at the lowest concentration quality control (QC) sample was 16% (difference from theoretical value) for 8-hydroxynevirapine, all others were closer to their known respective standards. Within- and between-day precisions were within 12% for quality control samples for all five metabolites. Repetitive thawing and freezing did not have an effect on any metabolite through a minimum of three cycles. Thawed samples, remaining in plasma for 4 h before extraction, were within 5% of theoretical value. Stability of the extracted samples on the autosampler at room temperature was evaluated for 48 h and was observed to be within 12% of a fresh analytical sample for 2-hydroxynevirapine and 3-hydroxynevirapine; other metabolites were within 6% of theoretical value. The utility of the analytical method was demonstrated using trough steady-state plasma samples collected from 48 patients in a hepatic impairment study.     

Determination of carboplatin in human plasma using HybridSPE-precipitation along with liquid chromatography-tandem mass spectrometry

The main purpose of this study was to develop and validate a rapid, specific, sensitive, and reliable LC-MS/MS-based bioanalytical method for the determination of carboplatin in human plasma. The optimal chromatographic behavior of carboplatin was achieved on a Biobasic SCX column (50 mm × 2.1 mm, 5 μm) using ion exchange chromatography. The total LC analysis time per injection was 2.6 min with a flow rate of 1.5 mL/min with a gradient elution. Optimization with regard to improving recovery and minimizing matrix effects using HybridSPE-precipitation (HybridSPE-PPT) has been evaluated under various extraction conditions. As a result, sample preparation via HybridSPE-PPT with 1% formic acid in acetonitrile in a 96-well format was applied for method validation and sample analysis and showed acceptable recovery of greater than 25% and negligible matrix effects. The method validation was conducted over the curve range of 2.00-2000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤4.8% relative standard deviation (RSD) and -13.2 to -3.6% relative errors (RE). The method was successfully applied to determine carboplatin in human plasma samples.     

Determination of derivatized l-alanosine in plasma by liquid chromatography-tandem mass spectrometry

A sensitive method was developed for quantitation of the cytotoxic antibiotic l-alanosine in human plasma. Alanosine was extracted from plasma by anion-exchange solid phase extraction, derivatized with dansyl chloride and analyzed by liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization in negative mode. Dansylation led to 50-fold improvement of method sensitivity over non-dansylated alanosine with a resulting 20 ng/ml limit of alanosine quantitation in plasma being achieved. The method was validated and applied for clinical studies of alanosine administered to cancer patients.     

Determination of telmisartan in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive method for the determination of the angiotensin II receptor antagonist, telmisartan, in human plasma has been developed. Telmisartan and the internal standard, diphenhydramine, were extracted from plasma using diethyl ether-dichloromethane (60:40, v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (85:15, v/v) adjusted to pH 4.5 after mixing with formic acid as mobile phase. Detection was carried out by multiple reaction monitoring on a Q-trap LC-MS/MS system with an ESI interface. The assay was linear over the range 0.5-600.0 ng/ml with a limit of quantitation of 0.5 ng/ml and a limit of detection of 0.05 ng/ml. Intra- and inter-day precision were <6.7% and <8.1%, respectively, and the accuracy was in the range 88.9-111.0%. The assay was applied to a pharmacokinetic study of telmisartan given as a single oral dose (80 mg) to healthy volunteers.     

Determination of dioscin in rat plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for dioscin in rat plasma was developed. Ginsenoside Rh2 was employed as an internal standard. Dioscin is a naturally occurring saponin present in many traditional Chinese medicinal plants. Dioscin was determined after the acetonitrile-mediated plasma protein precipitation. The mobile phase consisted of acetonitrile:10 mmol/l aqueous ammonium acetate (95:5, v:v), which was pumped at 0.8 ml/min. The analytical column (100 mm x 4.6 mm i.d.) was packed with Hypersil ODS material (5 microm). The standard curve was linear from 1 to 100 ng/ml. The assay was specific, accurate (percentage deviations from nominal concentrations were <10%), precise and reproducible (within- and between-day coefficients of variation <10%). Dioscin in rat plasma was stable over three freeze-thaw cycles and at ambient temperatures for 24 h. The utility of the assay was demonstrated by determining dioscin plasma concentrations in five rats for 120 h following a single oral gavage dose of 90 mg/kg.     

Determination of protodioscin in rat plasma by liquid chromatography-tandem mass spectrometry

Protodioscin (3-O-[alpha-L-rhamnopyranosyl-(1-->2)-{alpha-L-rhamnopyranosyl-(1-->4)}-beta-D-glucopyranosyl]-26-O-[beta-D-glucopyranosyl]-(25 R)-furost-5-ene-3 beta,26-diol) is a naturally occurring saponin present in many oriental vegetables and traditional medicinal plants, which has been associated with potent bioactivity. However, there is no specific and sensitive assay for quantitative determination of protodioscin in biological samples. We have established a rapid, sensitive and selective LC-ESI-MS/MS method to measure protodioscin in rat plasma and investigated the pharmacokinetics of protodioscin after intravenous administrations. Plasma samples were prepared after plasma protein precipitation, and a aliquot of the supernatant was injected directly onto an analytical column with a mobile phase consisted of acetonitrile-water-formic acid (80:20:0.1, v/v/v). Analytes were detected with a LC-ESI-MS/MS system in positive selected multiple reaction-monitoring mode. The lower limit of quantification (LLOQ) was 20.0 ng/mL and a linear range of 20-125,000 ng/mL. The intra- and inter-day relative standard deviation (R.S.D.) across three validation runs over the entire concentration range was <8.0%. Accuracy determined at three concentrations (50, 5000 and 50,000 ng/mL for protodioscin) ranged from 0.2 to 1.8% as terms of relative error (R.E.). Each plasma sample was chromatographed within 3.5 min. This LC-ESI-MS/MS method allows accurate, high-throughput analysis of protodioscin in small amounts of plasma.     

Determination of protease inhibitors using liquid chromatography-tandem mass spectrometry

A method for the analysis of six protease inhibitors and one metabolite has been developed and validated. Amprenavir, ritonavir, saquinavir, lopinavir, indinavir, nelfinavir, and an active metabolite of nelfinavir (M8) are quantitated using reversed-phase liquid chromatography coupled to tandem mass spectrometry, equipped with an electrospray ionization source (ESI-LC-MS-MS). The validation data presented here shows that the method allows the rugged analysis of these species from one aliquot. The evolution of complex drug interactions assessments and the clinical use of therapeutic drug monitoring for these antiretrovirals will be a potential immediate application of this method.     

Determination of solifenacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray tandem mass spectrometry method was developed and validated to quantitate solifenacin in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography performed on a pentafluorophenylpropylsilica column (50×4mm, 3μm particles), the mobile phase consisted of methanol - 100mM ammonium acetate containing 1% of formic acid (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 363→193 and 368→198 for solifenacin and the internal standard solifenacin-D(5), respectively. The lower limit of quantitation was 0.47ng/ml using 0.25ml of plasma and linearity was demonstrated up to 42ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 11% and inaccuracy did not exceed 11% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.