Selective and sensitive liquid chromatography-tandem mass spectrometry method for the determination of levonorgestrel in human plasma

A selective, sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of levonorgestrel in plasma was developed. An Applied Biosystems API 3000 triple quadrupole mass spectrometer set to multiple reaction monitoring (MRM) mode, using atmospheric pressure photospray ionisation (APPI) in the positive mode. Using 17-alpha-methyltestosterone as internal standard (IS), liquid-liquid extraction was followed by reversed phase liquid chromatography using a phenyl-hexyl column and tandem mass spectrometric detection. The mean recovery for levonorgestrel and 17-alpha-methyltestosterone was 99.5 and 62.9%, respectively. The method was validated from 0.265 to 130 ng levonorgestrel/ml plasma with the lower limit of quantification (LLOQ) set at 0.265 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS/MS) detection, allowing for a rapid (extraction and chromatography) and selective method for the determination of levonorgestrel in human plasma. The assay method was used in a pharmacokinetic study to quantify levonorgestrel in human plasma samples generated after administrating a single oral dose of 1.5 mg levonorgestrel to healthy female volunteers for up to five half lives. The total chromatographic runtime of this method was 5.0 min per sample, allowing for analysis of a large number of samples per batch.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of clonidine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of clonidine in human plasma. Clonidine was extracted from human plasma by using solid-phase extraction technique. Nizatidine was used as the internal standard. A Hypurity C18 (50 mm x 4.6 mm i.d., 5 microm particle size) column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection up to picogram levels with a total run time of 3.0 min only. The method was validated over the range of 50-2500 pg/mL. The absolute recoveries for clonidine (71.86%) and IS (69.44%) achieved from spiked plasma samples were consistent and reproducible.     

Validated method for the determination of risperidone and 9-hydroxyrisperidone in human plasma by liquid chromatography-tandem mass spectrometry

Since the first entry of risperidone on to the market in the early 1990s, investigation of the pharmacokinetic behaviour of the compound for which the availability of a bioanalytical method was a condition sine qua non, has received considerable attention. Most of the published methods, however, did not reach the level of sensitivity and selectivity which can be obtained today since the evolution of liquid chromatography-tandem mass spectrometry (LC-MS-MS) towards a routine technique in the bioanalytical laboratory. Therefore, we developed and validated a new LC-MS-MS method for the determination of risperidone and its active metabolite 9-hydroxyrisperidone in human plasma. This paper describes in detail the bioanalytical procedure and summarizes the validation results obtained. In addition, it focuses on the pitfalls one might encounter when developing similar assays. Despite the particular physicochemical characteristics of risperidone and 9-hydroxyrisperidone, the LC-MS-MS method enabled the quantification of both compounds down to 0.1 ng/ml. The method uses a sample preparation step by solid-phase extraction at pH 6 using a mixed-mode phase. In a short chromatographic run, separation of 9-hydroxyrisperidone from the minor metabolite 7-hydroxyrisperidone is achieved. Detection takes place by (turbo)ionspray tandem mass spectrometry in the positive ion mode. The validated concentration range is from 0.100 to 250 ng/ml, using 500 microliter of sample, with accuracy (bias) and precision (coefficient of variation) being below 15%. Although new developments in equipment will allow us to further improve and speed up the method, the assay reported can be used as a routine method to support a wide range of pharmacokinetic studies.     

Sensitive method for the determination of ibutilide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for determination of ibutilide in human plasma. The analyte and internal standard sotalol were extracted from plasma samples by liquid-liquid extraction, and separated on a C(18) column, using acetonitrile-water-10% butylamine-10% acetic acid (80:20:0.07:0.06, v/v/v/v) as the mobile phase. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via TurboIonSpray ionization (ESI). Linear calibration curves were obtained in the concentration range of 20-10,000 pg/ml, with a lower limit of quantitation of 10 pg/ml. The intra- and inter-day precision values were below 8% and accuracy was within +/-3% at all three QC levels. The method was utilized to support clinical pharmacokinetic studies of ibutilide in healthy volunteers following intravenous administration.     

Rapid determination of metformin in human plasma by liquid chromatography-tandem mass spectrometry method

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method is described for quantitation of metformin in human plasma. After a simple, one-step protein precipitation using acetonitrile, metformin and the internal standard diphenhydramine were chromatographed on a C(8) column and detected by tandem mass spectrometry. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The method has a chromatographic total run time of 3.4 min and was linear within the range 2-2000 ng/ml. Intra- and inter-day precision, expressed as the relative standard deviation (R.S.D.), ranged from 4.4 to 5.7% and from 1.3 to 2.8%, respectively. Assay accuracy was less than 1% in terms of %RE (relative error). The assay was used to evaluate the pharmacokinetics of metformin after an oral administration of multicomponent formulation containing 500 mg metformin and 2.5 mg glyburide to 20 healthy volunteers.     

Sensitive liquid chromatography-tandem mass spectrometry method for the determination of clarithromycin in human plasma

A sensitive method for the determination of clarithromycin in plasma is described, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Samples were prepared using liquid-liquid extraction and separated on a Supelco Discovery C18 column with a mobile phase consisting of acetonitrile, methanol and acetic acid. Detection was performed by a PE SCIEX API 2000 mass spectrometer in the multiple reaction monitoring (MRM) mode (LC-MS-MS) using TurbolonSpray ionization and monitoring the transition of the protonated molecular ion for clarithromycin at m/z 748.5 (M+1) to the predominant product ion of m/z 158.2. The mean recovery of clarithromycin was 87.3%, with a lower limit of quantification of 2.95 ng/ml when using 0.3-ml plasma. This high-throughput method was used to quantify 230 samples per day, and is sufficiently sensitive to be employed in pharmacokinetic studies.     

Sensitive method for the quantitative determination of bromocriptine in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive LC-MS-MS assay for the quantitative determination of bromocriptine has been developed and validated and is described in this work. The assay involved the extraction of the analyte from 1 ml of human plasma using a solid phase extraction on Oasis MCX cartridges. Chromatography was performed on a Symmetry C18 (2.1 mm x 100 mm, 3.5 microm) column using a mobile phase consisting of 25:75:01 acetonitrile-water-formic acid with a flow rate of 250 microl/min. The linearity was within the concentration range of 2-500 pg/ml. The lower limit of quantification was 2 pg/ml. This method has been demonstrated to be an improvement over existing methods due to its greater sensitivity and specificity.     

Determination of Tirofiban in human serum by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method with a rapid and simple sample preparation was developed and validated for the determination of Tirofiban in biological fluids. Tirofiban in serum samples was extracted and cleaned up by using an automated solid phase extraction method. An external calibration was used. The mass spectrometer was operated in the multiple reaction monitoring mode (MRM). A good linear response over the range of 2-200ng/ml was demonstrated. The accuracy for Tirofiban ranged from 94.8 to 110.8% within-day and from 103.0 to 104.7% between-day. The lower limit of quantification was 2ng/ml. This method is suitable for pharmacokinetic studies.     

Simple and sensitive liquid chromatography-tandem mass spectrometry method for determination of the S(+)- and R(-)-enantiomers of baclofen in human plasma and cerebrospinal fluid

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to determine the enantiomers of the muscle relaxant baclofen in human plasma and cerebrospinal fluid (CSF) has been developed. A commercially available ultrafiltration membrane is used to prepare the sample. A chiral CROWNPAK CR(+) stationary phase column is then used to perform complete resolution of the S(+)- and R(-)-enantiomers of baclofen. This method was used to analyze human plasma and CSF spiked with baclofen, and the calibration curves for both biologic samples were linear over a concentration range of 0.15-150 ng enantiomer/ml. The lower limit of quantification was 0.15 ng enantiomer/ml in both fluids. Finally, the method was tested with an artificial CSF as an alternative to authentic human CSF. The results showed that no matrix effects and no interfering peaks were observed using this artificial CSF.     

A sensitive liquid chromatography-tandem mass spectrometry method for the quantification of mometasone furoate in human plasma

A robust, rapid, selective and sensitive liquid chromatography-negative atmospheric pressure chemical ionization (LC-(APCI(-))-MS-MS) method has been developed for the quantification of mometasone furoate (MF) in human plasma utilizing a solid-phase extraction clean-up step and 13C-fluticasone propionate as internal standard. The intra- and inter-day coefficients of variation were < or = 15% and the lower limit of quantification (LLOQ) was 15 pg/ml. This method is ideally suited for pharmacokinetic investigations of low MF levels following inhalation of MF.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantitation of colchicine in human plasma

A rapid and sensitive method to determine colchicine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Colchicine and the internal standard (I.S.), tegafur, were extracted from the matrix with n-hexane:dichloromethane:isopropanol (300:150:15, v/v/v) and separated by reversed-phase high-performance liquid chromatography (HPLC) using formic acid:10 mM ammonium acetate:methanol (1:49:75, v/v/v) as the mobile phase in a run time of 2.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay was linear in the concentration range 0.050-10 ng/ml with intra- and inter-day precision (as relative standard deviation (R.S.D.)) of <2 and <7%, respectively. The method was applied to a pharmacokinetic study of colchicine in healthy volunteers given an oral dose of 2.0 mg.     

Determination of nimodipine in human plasma by a sensitive and selective liquid chromatography-tandem mass spectrometry method

A sensitive and highly selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to determine nimodipine in human plasma. The analyte and internal standard nitrendipine were extracted from plasma samples by n-hexane-dichloromethane-isopropanol (300:150:4, v/v/v), and chromatographed on a C(18) column. The mobile phase consisted of methanol-water-formic acid (80:20:1, v/v/v). Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source. The method has a limit of quantification of 0.24 ng/ml. The linear calibration curves were obtained in the concentration range of 0.24-80 ng/ml. The intra- and inter-day precisions were lower than 4.4% in terms of relative standard deviation (R.S.D.), and the accuracy ranged from 0.0 to 5.8% in terms of relative error (RE). This validated method was successfully applied for the evaluation of pharmacokinetic profiles of nimodipine tablets administered to 18 healthy volunteers.     

Extractionless and sensitive method for high-throughput quantitation of cetirizine in human plasma samples by liquid chromatography-tandem mass spectrometry

Following a single 10-mg oral dose of cetirizine dihydrochloride to 24 healthy volunteers, the analyte was quantified in human plasma. Protein precipitation using acetonitrile (ACN) was followed by reversed-phase liquid chromatography and tandem mass spectrometry. The MS/MS method was optimised using a PE Sciex API 2000 triple quadrupole mass spectrometer in selected reaction monitoring (SRM) mode, using electrospray with positive ionisation. Oxybutynin was used as the internal standard. The assay method represents a robust, high-throughput, highly specific and sensitive quantitative assay procedure, with 0.5 ng/ml being the lowest plasma concentration that could be reliably quantified. The procedure involves minimal sample preparation, and is well suited to clinical studies of the drug involving large numbers of generated samples. Pre-dose as well as post-dose samples up to and including 48 h were quantified, and the data generated were used to determine the pharmacokinetic profile of the drug.     

Quantitative determination of buagafuran in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of buagafuran in human plasma. The analyte was extracted from plasma samples with hexane after addition of isotopic internal standard and chromatographed on a RP-C(8) column. The mobile phase consisted of methanol-water (90:10, v/v) and the flow rate was 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.5-200 ng/mL. Inter- and intra-day precision (RSD%) were all within 15% and the accuracy (RE%) was equal or lower than 9.5%. The lower limit of quantitation (LLOQ) was 0.5 ng/mL. The extraction recovery was on average 38.1% and the detection was not affected by the matrix. The method was successfully applied to the pharmacokinetic study of buagafuran in healthy Chinese volunteers.     

Simple liquid chromatography-tandem mass spectrometry method for determination of novel anti-methicillin-resistant Staphylococcus aureus fluoroquinolone WCK 771 in human serum

A simple, rapid, specific, precise, accurate and sensitive method for determination of WCK 771 in human serum has been developed. The method uses high performance liquid chromatography with tandem mass spectrometric detection. Sample preparation involves protein precipitation method by addition of acetonitrile. Gatifloxacin was used as internal standard. The response was found to be linear from 0.312 to 40 microg/ml of serum with correlation coefficient greater than 0.99. Limit of detection and lower limit of quantification for WCK 771 was found to be 0.078 microg/ml and 0.312 microg/ml, respectively. The intra-day precision and accuracy from analysis of quality control (QC) samples at four concentrations was in the range of 2.36-2.58% and from 96.71 to 103.2%, respectively. The inter-day precision and accuracy from analysis of quality control samples at four concentrations was in the range of 3.14-6.82% and from 96.84 to 105.76%, respectively. WCK 771 was found to be stable for 24 h at auto-injector environment. WCK 771 was also found to be stable for 2h in serum at 25+/-3 degrees C and for 3 months at -20 degrees C. Mean absolute recovery at four different concentrations was 86.92% with standard deviation of 1.79. Throughput of the method is approximately one sample every 4 min. The method was also reproduced with monkey serum. The method was employed for estimation of drug serum levels during pre-clinical and clinical trials.     

Sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of meloxicam in human plasma

Meloxicam was quantified in human plasma after a single 15 mg oral dose of the drug was given to 26 healthy volunteers. An Applied Biosystems Sciex API 2000 triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray (TIS) in the positive ion mode, was used. Protein precipitation with acetonitrile was followed by C(18) reverse phase liquid chromatography and tandem mass spectrometry. The mean recovery for meloxicam was 92% with a lower limit of quantification of 8.96 ng/ml. Piroxicam was used as the internal standard. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometry (MS-MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of meloxicam in human plasma than has previously been described.     

Sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of stavudine in human plasma

A sensitive method for the determination of stavudine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from plasma with Waters, Sep-Pak Vac, 100 mg, tC(18) solid-phase extraction (SPE) columns. Chromatography was performed on a Supelco Discovery C(18), 5 microm, 150 x 2 mm column with a mobile phase consisting of ammonium acetate (0.01 M)-acetonitrile-methanol (800:100:100, v/v/v) at a flow-rate of 0.3 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for stavudine was 94% with a lower limit of quantification set at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS-MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of stavudine in human plasma than has previously been described.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of rivastigmine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of rivastigmine in human plasma. Rivastigmine was extracted from human plasma by using solid-phase extraction technique. Zolpidem was used as the internal standard. A Betabasic-8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 251.20-->206.10, 86.20 for rivastigmine and m/z 308.10-->235.10 for zolpidem. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatographic conditions and mass spectrometric detection that enables detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.2-20.0 ng/ml with a correlation coefficient > or =0.9988. The intra-run and inter-run precision and accuracy were within 10.0%. The overall recoveries for rivastigmine and zolpidem were 86.28% and 87.57%, respectively. The total run time was 2.0 min. The developed method was applied for the determination of the pharmacokinetic parameters of rivastigmine following a single oral administration of a 3 mg rivastigmine capsule in 20 healthy male volunteers.     

Rapid and sensitive determination of vinorelbine in human plasma by liquid chromatography-tandem mass spectrometry and its pharmacokinetic application

Vinorelbine is a semi-synthetic vinca alkaloid with demonstrated high activities against various types of advanced cancer. To support a clinical pharmacokinetic study, a simple, rapid and sensitive method to determine vinorelbine in human plasma was developed using reversed phase liquid chromatography (LC) coupled with electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). Vinorelbine and vinblastine (the internal standard) were extracted from human plasma by one-step liquid-liquid extraction (LLE) with methyl-t-butyl ether. The chromatographic separation was achieved on a Spursil polar-modified C(18) column (50 mm×2.1 mm, 3 μm, Dikma Technologies) with an isocratic mobile phase of a 75:25 (v/v) acetonitrile-4 mmol/L ammonium formate (pH 3.0) mixture at a flow-rate of 0.4 mL/min. The MS/MS detection was performed in the positive ion multiple reaction monitoring (MRM) mode by monitoring the precursor→product ion transitions at m/z 779.4→122.0 and m/z 811.3→224.2 for vinorelbine and the internal standard, respectively. The assay was validated in the range 0.1-200 ng/mL (r>0.997), the lowest level of this range being the lower limit of quantification (LLOQ) based on 50 μL of plasma. The intra- and inter-day precisions were within 6.0%, while the accuracy was within ±4.7% of nominal values. Detection and quantification of both analytes within 2 min make this method suitable for high-throughput analyses. The method was successfully applied to evaluate the systemic pharmacokinetics of vinorelbine after a 20-min intravenous infusion of 25 mg/m(2) of vinorelbine to patients with metastatic breast cancer.     

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.