Extended application of an LC-MS/MS method for the analysis of vesnarinone and its metabolites in human urine and dialysate fluid

Vesnarinone, a positive inotropic drug developed for congestive heart failure, and its metabolites (OPC-8230, OPC-18136, OPC-18137) were analyzed in human dialysate and urine (plus an additional metabolite: OPC-18692 in urine) samples using a modification to a previously published LC-MS/MS assay for the analysis of human plasma and urine samples. OPC-8192, a structural analogue of vesnarinone, was used as the internal standard. The analytes of interest were extracted from human dialysate or urine by a solid phase extraction method using a pre-conditioned C-18 extraction column. The analytes were then resolved by a 7 min gradient elution on a reverse phase high performance liquid chromatographic column. Vesnarinone and metabolites were detected on a PE/Sciex API III+Biomolecular Mass analyzer in MS/MS mode using a Turbo IonSpray interface. The linear range of quantitation in dialysate was 2.00-100.00 ng/ml for vesnarinone and 0.50-25.00 ng/ml for each metabolite. In urine, the linear range was of 0.50-25.00 microg/ml for vesnarinone and 0.10-5.00 microg/ml for the metabolites. This method was used to support the analysis of urine and dialysate samples from renally impaired patients who are on vesnarinone treatment.     

Determination of morphine and codeine in urine by gas chromatography—mass spectrometry

GC—MS is one of the recommended analytical techniques for the identification and confirmation of opiates in urine. A method for the qualitative detection and quantitation of codeine and morphine in urine samples by this technique has been developed. This method is also suitable for the detection of their main metabolites in urine: norcodeine and normorphine. It also allows the identification of 6-monoacetylmorphine in urine, which can be used as a confirmatory marker of heroine abuse.

The derivatized compounds are separated by capillary gas chromatography (GC) and identified by mass spectrometry (MS) in the selective ion monitoring acquisition mode (SIM).

The recoveries from urine at concentrations of 1000 ng ml⁻¹ are 72% for codeine and 80% for morphine. The method is linear in the range studied (0–1000 ng ml⁻¹) for codeine and morphine.     

Simultaneous separation and determination of active components in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0×150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]⁺ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4–140.0 μg ml⁻¹ for adenine, 0.6–117.5 μg ml⁻¹ for hypoxanthine, 0.5–128.5 μg ml⁻¹ for adenosine and 0.5–131.5 μg ml⁻¹ for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 μg ml⁻¹ for adenine, 0.6 and 0.2 μg ml⁻¹ for hypoxanthine, 0.5 and 0.1 μg ml⁻¹ for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.     

Determination of SR 49059 in human plasma and urine by LC-APCI/MS/MS

SR 49 059 ((2S 1-[(2R 3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide) is an orally active non-peptide vasopressin V_1a antagonist. A sensitive, selective, and robust LC-MS/MS method was developed to determine the plasma and urine concentrations of SR 49 059 in support of clinical studies. Plasma samples were prepared based on a rapid extraction procedure using Chem Elut™ cartridges. The extracted samples were analyzed on a C₁₈ HPLC column interfaced with a Finnigan TSQ 700 mass spectrometer. Positive atmospheric chemical ionization (APCI) was employed as the ionization source. The analyte and its internal standard (²H₆-SR 49 059) were detected by use of multiple reaction monitoring (MRM) mode. The plasma matrix had a calibration range 0.2−20 ng ml⁻¹, with within and between run accuracy and precision both less than 10%. The chromatographic run time was approximately 3 min. Urine samples were prepared based on a simple dilution with water, followed by analysis under the same conditions as plasma. The calibration range for urine matrix was 20–5000 ng ml⁻¹, with within and between run accuracy and precision less than 11%. The method has been successfully applied to the clinical sample analysis. The plasma assay was also evaluated on a Finnigan TSQ 7000 mass spectrometer. The performance based on precision and accuracy was virtually identical to that on the TSQ 700, with the exception of linearity in calibration curve (the TSQ 700 was linear, the TSQ 7000 was quadratic).     

An isocratic high-performance liquid chromatographic system for simultaneous determination of theophylline and its major metabolites in human urine

A simple and highly selective isocratic high-performance liquid chromatography method is presented for the simultaneous determination of theophylline and its major metabolites in human urine using β-hydroxyethyl theophylline as an internal standard. The method utilizes direct injection of diluted urine samples followed by separation and quantitation by reversed-phase isocratic elution and ultraviolet detection. The assay is accurate and reproducible with a sensitivity of 1 μg ml⁻¹ for theophylline and 0.5 μg ml⁻¹ for its metabolites. The assay was employed for the analysis of theophylline and its major metabolites in urine following the oral administration of theophylline to four healthy volunteers.     

Validation of a sensitive LC/MS/MS method for simultaneous quantitation of flupentixol and melitracene in human plasma

A sensitive method has been developed and validated, using LC/ESI-MS/MS, for simultaneous quantitation of flupentixol and melitracen—antidepressant drugs, in human plasma. The quantitation of the target compounds was determined in a positive ion mode and multiple reaction monitoring (MRM). The method involved a repeated liquid–liquid extraction with diethyl ether and analytes were chromatographed on a C₈ chromatographic column by elution with acetonitrile–water–formic acid (36:64:1, v/v/v) and analyzed by tandem mass spectrometry. The method was validated over the concentration ranges of 26.1–2090 pg/ml for flupentixol and 0.206–4120 ng/ml for melitracen. The correlation coefficients of both analyst were >0.998 for six sets of calibration curves. The recovery was 60.9–75.1% for flupentixol, melitracen and internal standard. The lower limit of quantitation (LLOQ) detection was 26.1 pg/ml for flupentixol and 0.206 ng/ml for melitracen. Intra- and inter-day precision of the assay at three concentrations were 2.15–5.92% with accuracy of 97.6–103.0% for flupentixol and 0.5–6.36% with accuracy of 98.7–101.7% for melitracen. Stability of compounds was established in a battery of stability studies, i.e., bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for bioequivalence study of flupentixol and melitracen in healthy human male volunteers.     

High-throughput quantitation of nefazodone and its metabolites in human plasma by high flow direct-injection LC-MS/MS

A rapid, selective and sensitive high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method coupled with high flow direct-injection on-line extraction has been developed and validated for the simultaneous quantitation of nefazodone and its three active metabolites, hydroxynefazodone, triazole-dione (BMS-180492) and m-chlorophyenylpiperazine (mCPP) in human plasma. The method utilized d₇-nefazodone, d₇-hydroxynefazodone, d₄-BMS-180492 and d₄-mCPP as internal standards (IS). The plasma samples were injected into the LC–MS/MS system after simply adding the internal standard solution and centrifuging. The required extraction and chromatographic separation of the analytes were achieved on an Oasis^® HLB column (on-line extraction column, 1 mm × 50 mm, 30 μm) and a conventional Luna C8 column (analytical column, 4.6 mm × 50 mm, 5 μm). Detection was by positive ion electrospray tandem mass spectrometry. The total analysis run time for each sample was 2 min, which included the time needed for on-line extraction, chromatographic separation and LC–MS/MS analysis. The assay was validated for each analyte and the concentrations ranged from 2.0 to 500 ng/ml for nefazodone, hydroxynefazodone and mCPP and from 4.0 to 1000 ng/ml for BMS-180492, respectively. The assay was used for the high-throughput sample analysis of thousands of pharmacokinetic study samples and was proven to be rapid, accurate, precise, sensitive, specific and rugged.     

A study of matrix effects on an LC/MS/MS assay for olanzapine and desmethyl olanzapine

The purpose of this research project was to investigate potential matrix effects of anticoagulant and lipemia on the response of olanzapine, desmethyl olanzapine, olanzapine-D₃ and desmethyl olanzapine-D₈ in an LC/MS/MS assay. Blank human serum and sodium heparin, sodium citrate, and K₃EDTA plasma with various degrees of lipemia were fortified with olanzapine, desmethyl olanzapine, olanzapine-D₃ and desmethyl olanzapine-D₈. Six replicates of each sample were extracted using Waters Oasis^® MCX cartridges and analyzed using electrospray LC/MS/MS. The analytes were separated on a Phenomenex LUNA phenyl hexyl, 2 mm×50 mm, 5 μm, analytical column and a gradient rising from 2 to 85% mobile phase B. Mobile phase A consisted of acetonitrile–ammonium acetate (20 mM) (52:48 v/v) and mobile phase B was formic acid–acetonitrile (0.1:100 v/v). Ion suppression was investigated through post column infusion experiments. The degree of lipemia of each sample, indicated by turbidity, was ranked into categories from least to greatest and used for statistical analyses. The results from analysis of variance testing indicated that lipemia, anticoagulant and their interaction significantly influenced mass spectral matrix effects and extraction matrix effects. Differential behavior between the analytes and labeled internal standards contributed to variability. The most significant source of variability however, was ion suppression due to co-eluting matrix components.     

Two-dimensional LC–MS/MS determination of antiretroviral drugs in rat serum and urine

A simple, rapid, reliable and highly sensitive on-line two-dimensional reversed-phase liquid chromatography–tandem mass spectrometric (2D-LC/MS/MS) method to determine antiretroviral drugs viz., abacavir (ABC), nevirapine (NVP) and indinavir (IDV) in rat serum and urine was developed and validated. The analytes were extracted on-line from rat serum and urine by a restricted access material (RAM) column and back-flushed into the reversed-phase C₁₈ column for separation by LC. Detection was carried out by ESI-MS/MS. The developed method showed good selectivity, accuracy and precision for quantification of the antiretroviral drugs in rat serum and urine. Quantification limits for abacavir and nevirapine were 4.0 ng ml⁻¹, whereas for indinavir 4.7 ng ml⁻¹. The calibration graphs were linear in the range of 4–50 ng ml⁻¹for abacavir, nevirapine and indinavir. The method was successfully applied to study the pharmacokinetics of antiretroviral in rats.     

The determination of non-steroidal anti-inflammatory drugs by GC—MS—MS in equine urine

Results are given for a more sensitive screening procedure for non-steroidal anti-inflammatory drugs using GC—MS—MS. By monitoring a selected characteristic reaction for each drug very low detection limits are reached even in a difficult biological matrix such as equine urine. Detection down to 5 ng ml⁻¹ for ibuprofen, ibufenac, alclofenac, fenoprofen, ketoprofen, naproxen and diclofenac is possible in contrast to the 0.5 μg ml⁻¹ limit for normal GC—MS detection. Examples are given of real positive cases for diclofenac and ibuprofen.     

N-deethylation and N-oxidation of etamiphylline: identification of etamiphylline-N-oxide in greyhound urine by high performance liquid chromatography-mass spectrometry

Millophyline-V^®, (etamiphylline camsylate) was administered intramuscularly to two racing greyhounds at a dose of 10 mg kg⁻¹. Unhydrolysed pre- and post-administration urine samples were extracted using mixed mode solid phase extraction (SPE) cartridges, the basic isolates derivatised as trimethylsilyl ethers and analysed by positive ion electron ionisation gas chromatography–mass spectrometry (GC/EI+/MS). The parent drug and one metabolite, N-desethyletamiphylline, were detected in urine for up to 72 h. For semi-quantification, urine samples were extracted on-line using a Prospekt sample handler. The analytes retained on the C₂ SPE cartridge were eluted by the mobile phase directly on to the analytical high performance liquid chromatography column and analysed by positive ion atmospheric pressure chemical ionisation (LC/APCI+) MS in the multiple selective-ion recording mode. A major peak containing both ions (m/z) 280 and (m/z) 252 was observed. Full scan LC/APCI+/MS of the unknown indicated that the ion at (m/z) 280 was formed by the loss of an oxygen atom [MH⁺ → (MH⁺ − O)]. Samples were analysed by positive ion electrospray ionisation LC/MS on two different instruments and the unknown compound was identified as an N-oxide of the tert. nitrogen atom of the 2-(diethylamino)ethyl substituent on N₇ of the theophylline nucleus. This compound has not been reported previously either as an in vivo or in vitro metabolite of etamiphylline in any species. Thermal decomposition of the N-oxide could lead to an increase the detection period of the parent drug during routine GC/MS screening of post-competition greyhound urine samples.     

Liquid chromatographic determination of hydralazine in human plasma with 2-hydroxy-1-naphthaldehyde pre-column derivatization

A selective and sensitive high-performance liquid chromatographic method is described for determination of hydralazine and its metabolites in human plasma. The method involves pre-column derivatization with 2-hydroxy-1-naphthaldehyde at pH 1.2. The reaction product and Methyl Red used as internal standard are extracted into dichloromethane and chromatographed in the reversed-phase mode on an ODS-2 column using acetonitrile—aqueous triethylamine phosphate buffer (80:20, v/v) at pH 3 as eluent.

The plasma calibration curve of hydralazine is linear in the concentration range 10–500 ng ml⁻¹. The detection limit is 1 ng ml⁻¹ and the relative standard deviation is <2.4. In vivo pharmacokinetics of hydralazine in two volunteers after oral administration of 50 mg of the drug is studied using the proposed LC method.     

Determination of 5-fluorouracil in human plasma by a simple and sensitive reversed-phase HPLC method

A simple and sensitive reversed-phase HPLC method with UV detection was developed and validated for the quantitation of 5-fluorouracil (5-FU) in human plasma. After acidification and salting out, 5-FU was extracted into ethyl acetate and back-extracted into a basic buffer. The extract was adjusted to neutral pH before being injected onto the HPLC column. 5-FU was separated from the matrix components on a YMC ODS-AQ column at 40°C using an aqueous mobile phase of 10 mM potassium phosphate at pH 5.5. A linear gradient of 0–25% methanol wash eluted late peaks, maintained column performance, and increased column stability. The run time was 20 min. The linear range was 25–300 ng ml⁻¹ (r²>0.999). The limit of quantitation was 25 ng ml⁻¹, with a signal-to-noise ratio of 23:1. Interday precision and accuracy of quality control samples were 6.2–8.4%, relative standard deviation and −0.1– + 1.9% relative error.     

Specific and sensitive determination of digoxin and metabolites in human serum by high performance liquid chromatography with cyclodextrin solid-phase extraction and precolumn fluorescence derivatization

A precolumn fluorescence derivatization high performance liquid chromatographic method has been developed for the simultaneous determination of digoxin and its metabolites digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside digoxigenin, and dihydrodigoxin (20-R and 20-S epimers) in human serum. Digoxin and its metabolites were extracted from serum samples (containing digitoxin as internal standard) with a cyclodextrin solid-phase extraction (SPE) column. Fluorescent derivatives were formed by reaction of the analytes with 1-naphthoyl chloride in the presence of 4-dimethylaminopyridine under a nitrogen atmosphere in a glove box with controlled relative humidity (26% r.h. or less). The derivatives were isolated using cyclodextrin and Cl SPE columns sequentially, and determined by HPLC using silica column separation and fluorescence detection. Calibration curves were linear over the concentration range from 0.25 to 4.0 ng ml⁻¹. Recoveries of digoxin and its metabolites from serum ranged from 62 to 86%, and coefficients of variation from repetitive analyses ranged from 6.9 to 20.9% and from 5.8 to 12.2% at 0.5 ng ml⁻¹ and 2.0 ng ml⁻¹, respectively. This method has been shown capable of specifically determining digoxin and its major metabolites in serum, and has been successfully used in the determination of digoxin and its metabolites in serum samples collected from patients undergoing digoxin therapy. This method thus permits the investigation of digoxin metabolism and pharmacokinetics after the administration of commercial dosage forms.     

Development and validation of two chromatographic methods for the quantification of E-6087 and one of its metabolites, E-6132, in rat plasma

E-6087 is a nonsteroidal anti-inflammatory compound under development that selectively inhibits cyclooxygenase-2. In vitro studies have shown that one of its metabolites, E-6132, also inhibits this enzyme. Due to chromatographic reasons, two reverse phase HPLC methods were developed and validated in order to elucidate which compound is responsible for the pharmacological activity in vivo. Chromatographic separation of E-6087 was achieved using acetonitrile–phosphate buffer (pH 2.5; 25 mM) (60:40, v/v) as mobile phase and two 4.6×150 mm×5 μm Inertsil ODS-2 columns. For E-6132, two Inertsil ODS-3 columns and 52% of acetonitrile were used instead. Internal standards and fluorescence detection differed between both methods. The same on-line solid-phase extraction method was used. Mean retention times for E-6087 and E-6132 were 15.2 (±1.3) and 36.1 (±0.6) min, respectively. The methods were selective and linear over the concentration range of 10–500 ng ml⁻¹ (r²>0.996) for E-6087 and 5–200 ng ml⁻¹ (r²>0.997) for E-6132. The limits of quantitation were 10 ng ml⁻¹ (E-6087) and 5 ng ml⁻¹ (E-6132) with a precision and accuracy <16% (E-6087) and <11% (E-6132). Mean recoveries from plasma were 43.2–61.9% (E-6087) and 60.4–65.2% (E-6132). For both compounds, both inter-assay and intra-assay precision and accuracy were within acceptable limits (<15%). As an example of the suitability of these methods, the results from a pharmacokinetic study are reported. After single oral administration of 5 mg kg⁻¹ of E-6087 to rats, plasma concentrations of E-6087 at peak time were higher than those of E-6132, suggesting that activity is mainly due to E-6087.     

Determination of fentanyl in human plasma and fentanyl and norfentanyl in human urine using LC-MS/MS

Fentanyl, a potent analgesic drug, has traditionally been used intravenously in surgical or diagnostic operations. Formulations with fentanyl in oral transmucosal delivery system and in transdermal depot-patch have also been developed against breakthrough pain in cancer patients. In this report, LC–MS/MS methods to determine fentanyl in human plasma as well as fentanyl and its main metabolite, norfentanyl, in human urine are presented together with validation data. The validation ranges were 0.020–10.0 and 0.100–50.0 ng/ml for fentanyl in plasma and urine, respectively, and 0.102–153 ng/ml for norfentanyl in urine.

Liquid–liquid extraction of the compounds fentanyl, norfentanyl and the deuterated internal standards, fentanyl-d₅ and norfentanyl-d₅ from the matrixes was applied and separation was performed on a reversed phase YMC Pro C₁₈-column followed by MS/MS detection with electrospray in positive mode. The inter-assay precision (CV%) was better than 4.8% for fentanyl in plasma and 6.2% and 4.7% for fentanyl and norfentanyl, respectively, in urine.

The ruggedness of the methods, selectivity, recovery, effect of dilution and long-term stability of the analytes in plasma and urine were investigated. Effect of haemolysis and stability of fentanyl in blood samples were also studied.

The methods have been applied for the determination of fentanyl in plasma samples and fentanyl/norfentanyl in urine samples taken for pharmacokinetic evaluation after a single intra-venous (i.v.) dose of 75 μg fentanyl.     

A highly sensitive and selective method for the determination of Leukotriene B4 in human plasma by negative ion chemical ionization/gas chromatography/tandem mass spectrometry

We have developed a highly sensitive and highly selective method for the determination of Leukotriene B₄ (LTB₄) in human plasma using negative ion chemical ionization/gas chromatography/tandem mass spectrometry (NICI/GS/MS/MS) analysis. The developed method was summarized as follows. Deuterated LTB₄ (d₄-LTB₄) was added to human plasma samples as an internal standard, and samples were extracted by a Sep-pak C18 column. Extracted LTB₄ was derivatized into the pentafluorobenzyl ester of bis-trimethylsilyl ether (PFB-TMS-LTB₄) and quantified on the basis of selected reaction monitoring (SRM) at m/z 299 of [M-PFB-2TMSOH]⁻ by NICI/GC/MS/MS analysis, which was the product ion of [M-PFB]⁻. The detection limit for the quantification of LTB₄ in human plasma was 10 pg ml⁻¹, sufficiently sensitive to determine the concentrations of endogenous LTB₄ in human plasma. The plasma level of LTB₄ measured in healthy male volunteers was 33.85 ± 33.91 pg ml⁻¹ (mean ± S.D. in six volunteers). The technique of MS/MS used in this method offers much greater sensitivity and selectivity than single-stage mass spectrometry. The developed method showed good reproducibility with a simple and rapid extraction procedure, and would be useful for examining the relationship between various disease states and the levels of LTB₄ in biological fluids.     

Simultaneous analysis of methylprednisolone, methylprednisolone succinate, and endogenous corticosterone in rat plasma

A reversed-phase HPLC method is reported for simultaneous quantitation of methylprednisolone (MP), MP succinate (MPS), and endogenous corticosterone (CST) in plasma of rats. Additionally, the 11-keto metabolite of MP (methylprednisone, MPN) is resolved from the other analytes. After addition of internal standard (triamcinolone acetonide; IS) and an initial clean up step, the analytes of interest are extracted into methylene chloride. The steroids are then resolved on a reversed-phase polymer column using a mobile phase of 0.1 M acetate buffer (pH 5.7): acetonitrile (77:23) which is pumped at a flow rate of 1.5 ml min⁻¹. Sample detection was accomplished using an UV detector at a wavelength of 250 nm. All the five components (MPS, MP, MPN, CST and IS) were baseline resolved from each other and other components of plasma. Linear relationships were found between the steroids: IS peak area ratios and plasma concentrations in the range of 0.1–4 μg ml⁻¹ for MP and MPS and 0.1–1.0 μg ml⁻¹ for MPN and CST. The assay is accurate as intra- and inter-run error values were <±8% for all the components. Further, the intra- and inter-run CVs of the assay were <16% at all the concentrations and for all the components. The application of the assay was demonstrated after the injection of a single 5 mg kg⁻¹ (MP equivalent) dose of MPS or a macromolecular prodrug of MP to rats.     

Fully automated methods for the determination of hydrochlorothiazide in human plasma and urine

LC assays utilizing fully automated sample preparation procedures on Zymark PyTechnology™ Robot and BenchMate™ Workstation for the quantification of hydrochlorothiazide (HCTZ) in human plasma and urine have been developed. After aliquoting plasma and urine samples, and adding internal standard (IS) manually, the robot executed buffer and organic solvent addition, liquid—liquid extraction, solvent evaporation and on-line LC injection steps for plasma samples, whereas, BenchMate™ performed buffer and organic solvent addition, liquid—liquid and solid-phase extractions, and on-line LC injection steps for urine samples. Chromatographic separations were carried out on Beckman Octyl Ultrasphere column using the mobile phase composed of 12% (v/v) acetonitrile and 88% of either an ion-pairing reagent (plasma) or 0.1% trifluoroacetic acid (urine). The eluent from the column was monitored with UV detector (271 nm). Peak heights for HCTZ and IS were automatically processed using a PE-Nelson ACCESS*CHROM laboratory automation system. The assays have been validated in the concentration range of 2–100 ng ml⁻¹ in plasma and 0. 1–20 μg ml⁻¹ in urine. Both plasma and urine assays have the sensitivity and specificity necessary to determine plasma and urine concentrations of HCTZ from low dose (6.25/12.5 mg) administration of HCTZ to human subjects in the presence or absence of losartan.