Liquid chromatographic–tandem mass spectrometric method for the determination of the neuraminidase inhibitor zanamivir (GG167) in human serum

An LC–MS–MS method for the analysis of the neuraminidase inhibitor, zanamivir, in human serum is described. Zanamivir was extracted from protein precipitated human serum samples using Isolute SCX solid-phase extraction cartridges and analysed using reversed-phase chromatography with TurboIonSpray atmospheric pressure ionisation followed by mass spectrometric detection. The method uses a stable isotope internal standard, is highly specific and sensitive for a compound of this type and has been used for the analysis of human serum and urine samples from clinical studies. The method was extended to the analysis of serum and plasma samples from pre-clinical studies involving the rat, ferret and cell culture media. The method has been shown to be robust and valid over a concentration range of 10–5000 ng/ml using a 0.2-ml sample volume. The main advantages of this method compared to earlier procedures are primarily specificity, sensitivity, ease of sample preparation, small sample volume and short analysis time (ca. 5 min).     

Rapid and selective method for norepinephrine in rat urine using reversed-phase ion-pair high-performance liquid chromatography-tandem mass spectrometry

A rugged, high-throughput HPLC–MS–MS-based method, suitable for quantitation of norepinephrine (NE) in urine, has been developed. A rapid, batch-mode procedure utilizes alumina to isolate NE and its deuterated internal standard from urine. After release of NE, using dilute formic acid, samples are analyzed by isocratic reversed-phase ion-pair HPLC, with electrospray ionization (ESI) and MS–MS detection. The ion-pair reagent, heptafluorobutyric acid, is compatible with the ESI interface and permits use of mobile phases with relatively high methanol content, enhancing ESI sensitivity. Furthermore, no significant drop in sensitivity is observed throughout more than 15 h of instrument operation. The selectivity of this approach permitted simplification of the extraction procedure and reduced run times (under 4 min), making single batch-run sizes of more than 200 samples practical. The lower limit of quantitation is 5 ng per 0.5 ml sample, with analytical recoveries of 97–100% and overall method precision of better than 4% relative standard deviation verified up to 500 ng ml⁻¹. This method was initially applied to study the diurnal rhythm in sympathetic nervous system activity of spontaneously hypertensive rats.     

Simple and reliable high-performance liquid chromatography fluorimetric procedure for the determination of amphetamine-derived designer drugs

The paper describes a HPLC–fluorimetric procedure for the determination of methylenedioxyamphetamine, methylenedioxymethamphetamine, methylenedioxyethamphetamine and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine in urine, serum, saliva and street samples, that features interesting advantages over other procedures previously described. The method requires a very small sample volume (100 μl) and no extraction, lacks matrix effect, and is not time consuming. Linearity was in the range 50–1000 ng/ml regardless of matrix. Sensitivity and detection limit were 50 ng/ml and 10 ng/ml, respectively, but they may reach 10 ng/ml and 2 ng/ml if a slight modification is introduced in the procedure. Intra- and inter-day precision were always within 5% and 8%, respectively. Recovery was satisfactory for all matrices. The described procedure could be successfully used for clinical, epidemiological and forensic applications.     

Determination of clenbuterol in bovine urine using gas chromatography–mass spectrometry following clean-up on an ion-exchange resin

A gas chromatographic–mass spectrometric method was developed for the determination of residues of clenbuterol in bovine urine. The method involves a simple cation-exchange clean-up and concentration of clenbuterol in the acidified urine, followed by ethyl acetate extraction. The analyte is determined as the di-trimethylsilyl derivative and quantitated against an internal standard of penbutolol. Using a 5-ml sample of urine, a detection limit of 0.07 ng/ml can be achieved with recoveries close to 100% for fortification levels of 0.2 and 1 ng/ml. By increasing the sample volume to 50 ml, a detection limit below 0.01 ng/ml was achievable with recovery averaging 70%. The coefficient of variation of the assay ranged from 15% at 0.01 ng/ml (50-ml sample) to 6% at 1 ng/ml (5-ml sample). It was demonstrated that the method can detect the presence of clenbuterol in bovine urine at sub-ppb (ng/ml) levels using low resolution GC–MS with electron impact (EI) ionization.     

Determination of risedronate in human urine by column-switching ion-pair high-performance liquid chromatography with ultraviolet detection

An HPLC assay for the determination of risedronate in human urine was developed and validated. Risedronate and the internal standard were isolated from 5-ml urine samples in a two-part procedure. First, the analytes were precipitated from urine along with endogenous phosphates as calcium salts by the addition of CaCl(2) at alkaline pH. The precipitate was then dissolved in 0.05 M ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and subjected to ion-pair solid-phase extraction using a Waters HLB cartridge (1 ml, 30 mg) with 1-octyltriethylammonium phosphate as the ion-pair reagent. Following extraction, the analytes were initially separated from the majority of co-extracted endogenous components on a Waters X-Terra RP18 (4.6 x 50 mm, 3.5 microm) column. The effluent from the X-Terra was "heart-cut" onto a Phenomenex Synergi Polar RP (4.6 x 150 mm, 4 microm) column for final separation. UV detection (lambda=262 nm) was used to quantitate risedronate in the concentration range of 7.5-250 ng/ml. Mean recovery was 83.3% for risedronate and 86.5% for the internal standard. The intra-day precision of the assay, as assessed by replicate (n=5) standard curves, was better than 6% RSD for all points on the standard curve. Within-day accuracy for the standards ranged from 96.3 to 106.1% of nominal. Inter-day precision for quality controls assayed over a 3-week period was better than 5%, while inter-day accuracy was within 90% of nominal. The assay was employed to analyze samples collected during a clinical pharmacokinetics study.     

Analysis of aplidine (dehydrodidemnin B), a new marine-derived depsipeptide, in rat biological fluids by liquid chromatography–tandem mass spectrometry

Aplidine (dehydrodidemnin B) is a new marine-derived depsipeptide with a powerful cytotoxic activity, which is under early clinical investigation in Europe and in the US. In order to investigate the pharmacokinetic properties of this novel drug, an HPLC–tandem mass spectrometry method was developed for the determination of aplidine in biological samples. Didemnin B, a hydroxy analogue, was used as internal standard. After protein precipitation with acetonitrile and extraction with chloroform, aplidine was chromatographed with a RP octadecylsilica column using a water–acetonitrile linear gradient in the presence of formic acid at the flow-rate of 500 μl/min. The method was linear over a 5–100 ng/ml range (LOD=0.5 ng/ml) in plasma and over a 1.25–125 ng/ml range (LOD=0.2 ng/ml) in urine with precision and accuracy below 14.0%. The intra- and inter-day precision and accuracy were below 12.5%. The extraction procedure recoveries for aplidine and didemnin B were 69% and 68%, respectively in plasma and 91% and 87%, respectively in urine. Differences in linearity, LOQ, LOD and recoveries between plasma and urine samples seem to be matrix-dependent. The applicability of the method was tested by measuring aplidine in rat plasma and urine after intravenous treatment.     

Determination of paclitaxel and metabolites in mouse plasma,tissues, urine and faeces by semi-automated reversed-phase high-performance liquid chromatography

We have developed and validated a sensitive and selective assay for the quantification of paclitaxel and its metabolites 6α,3′-p-dihydroxypaclitaxel, 3′-p-hydroxypaclitaxel and 6α-hydroxypaclitaxel in plasma, tissue, urine and faeces specimens of mice. Tissue and faeces were homogenized (approximately 0.1–0.2 g/ml) in bovine serum albumin (40 g/I) in water, and urine was diluted (1:5, v/v) in blank human plasma. Sample pretreatment involved liquid-liquid extraction of 200–1000 μl of sample with diethyl ether followed by automated solid-phase extraction using cyano Bond Elut column. 2′-Methylpaclitaxel was used as internal standard. The overall recovery of the sample pretreatment procedure ranged from 76 ot 85%. In plasma, the lower limit of detection (LOD) and the lower limit of quantitation (LLQ) are 15 and 25 ng/ml, respectively, using 200 μl of sample. In tissues, faeces and urine the LLQs are 25–100 ng/g, 125 ng/g and 25 ng/ml, respectively, using 1000 μl (faeces: 200 μl) of homogenized or diluted sample. The concentrations in the various biological matrices, for validation procedures spiked with known amounts of the test compounds, are read from calibration curves constructed in blank human plasma in the range 25–100 000 ng/ml for paclitaxel and 25–500 ng/ml for the metabolites. The accuracy and precision of the assay fall within the generally accepted criteria for bio-analytical assays.     

Determination of pamidronate in urine by ion-pair liquid chromatography after derivatization with 1-naphthylisothiocyanate

A sensitive method for the determination of pamidronate disodium [(3-amino-1-hydroxypropylidene)bisphosphonate, APD] in urine has been developed and validated. The procedure involves a triple co-precipitation with calcium phosphate, solid-phase extraction on a quaternary ammonium column, derivatization with 1-naphthylisothiocyanate and ion-pair liquid–liquid extraction. From the two reaction products, naphthylthiocarbamyl-APD is converted into the other, naphthylcarbamyl-APD, by an oxidative desulphuration with hydrogen peroxide prior to analysis by ion-pair HPLC and fluorescence detection at 285/390 nm. The method has a coefficient of variation of 7% for the intra-assay precision of 99 ng ml⁻¹ APD and 11% for the inter-assay precision. The lower limit of quantification is 3 ng ml⁻¹ APD in 2.5 ml of human urine.     

Simultaneous determination of flupirtine and its major active metabolite in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective and sensitive HPLC–tandem mass spectrometry method was developed and validated for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The analytes and internal standard diphenhydramine were extracted from plasma samples by liquid–liquid extraction, and chromatographed on a C₁₈ column. The mobile phase consisted of acetonitrile–water–formic acid (60:40:1, v/v/v), at a flow rate of 0.5 ml/min. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method has a limit of quantitation of 10 ng/ml for flupirtine and 2 ng/ml for D-13223, using 0.5-ml plasma sample. The linear calibration curves were obtained in the concentration range of 10.0–1500.0 ng/ml for flupirtine and 2.0–300.0 ng/ml for D-13223. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples was less than 7.2% for flupirtine and D-13223. The accuracy as determined from QC samples was less than 5% for the analytes. The overall extraction recoveries of flupirtine and D-13223 were determined to be about 66% and 78% on average, respectively. The method was applied for the evaluation of the pharmacokinetics of flupirtine and active metabolite D-13223 in volunteers following peroral administration.     

Determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine using high-performance liquid chromatography with fluorescence detection

A method for the determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine is described. Plasma and urine samples were extracted using 3M Empore extraction disk cartridges in the C₁₈ and MPC (mixed-phase cation-exchange) formats, respectively. The extract was analyzed using HPLC with fluorescence detection (ex 248 nm, em 300 nm), and included a column switching procedure to reduce run-time. The assay was linear in the concentration range 5 to 1000 ng/ml when 1-ml aliquots of plasma and urine were extracted. Recoveries of L-756 423 were greater than 84% over the calibration curve range using the described sample preparation procedures. Intra-day precision and accuracy for this assay was less than 9% RSD and within 7%, respectively. Inter-day variabilities for the plasma (n=17) and urine (n=10) were less than 5% and 3% for low (15 ng/ml) and high (750 ng/ml) quality control samples. Bovine serum albumin (0.5%) was used as an additive to urine to prevent precipitation of L-756 423 during the storage of clinical samples. The assay was used in support of human clinical trials.     

Quantitative liquid chromatographic–tandem mass spectrometric determination of reserpine in FVB/N mouse plasma using a “chelating” agent (disodium EDTA) for releasing protein-bound analytes during 96-well liquid–liquid extraction

A sensitive, specific, accurate and reproducible analytical method employing a divalent cation chelating agent (disodium EDTA) for sample treatment was developed to quantitate reserpine in FVB/N mouse plasma. Samples pretreated with 40 μl of 2% disodium EDTA in water were extracted by a semi-automated 96-well liquid–liquid extraction (LLE) procedure to isolate reserpine and a structural analog internal standard (I.S.), rescinnamine, from mouse plasma. The extracts were analyzed by turbo ionspray liquid chromatography–tandem mass spectrometry (LC–MS–MS) in the positive ion mode. Sample preparation time for conventional LLE was dramatically reduced by the semi-automated 96-well LLE approach. The assay demonstrated a lower limit of quantitation of 0.02 ng/ml using 0.1-ml plasma sample aliquots. The calibration curves were linear from 0.02 to 10 ng/ml for reserpine. The intra- and inter-assay precision of quality control (QC) samples ranged from 1.75 to 10.9% for reserpine. The intra- and inter-assay accuracy of QC samples ranged from −8.17 to 8.61%. Reserpine and the I.S. were found to be highly bound to FVB/N mouse plasma protein. This is the first report of disodium EDTA employed as a special protein-bound release agent to recover protein-bound analytes from plasma. These matrix effects and the effects of pH in the HPLC mobile phase on the sensitivities of LC–MS–MS are discussed in this paper.     

Automated high-performance liquid chromatographic method for the determination of a neuraminidase inhibitor (GG167) in human serum by pre-column fluorescence derivatisation using benzoin

A pre-column fluorescence derivatisation high-performance liquid chromatographic method for the analysis of a neuraminidase inhibitor, GG167, in human serum is described. GG167 was extracted from serum samples using Bond Elut SCX solid-phase extraction cartridges, followed by derivatisation with benzoin prior to reversed-phase chromatography with fluorescence detection. This method has been automated using a Zymark robot and used in the analysis of human serum samples from clinical studies. The method has been shown to be valid over a concentration range of 10–800 ng/ml using a 1-ml sample volume.     

Semi-automatic liquid chromatographic analysis of pamidronate in serum and citrate plasma after derivatization with 1-naphthylisothiocyanate

The semi-automatic method for the determination of the bisphosphonate pamidronate in serum and citrate plasma involves a manual protein precipitation with trichloroacetic acid and a manual coprecipitation of the bisphosphonate with calcium phosphate, followed by an automated solid-phase extraction on anion-exchange columns. After off-line evaporation of the extract under nitrogen and reconstitution in water, the automatic procedure is continued by automatic derivatization with 1-naphthylisothiocyanate, ion-pair liquid–liquid extraction and a treatment with hydrogen peroxide, prior to analysis by ion-pair HPLC and fluorescence detection at 285/390 nm. The intra- and inter-day precisions are 1.3 and 7%, respectively, for a standard of 100 ng ml⁻¹ pamidronate in serum; the average accuracy for this standard is 107%. The lower limit of quantification is 20 ng ml⁻¹ pamidronate in 1 ml of human serum.     

Semi-automatic liquid chromatographic analysis of pamidronate in urine after derivatization with 1-naphthylisothiocyanate

An existing sensitive chromatographic assay for pamidronate in urine has considerably been automated. Using the same sample processor, the solid-phase extraction (SPE) was automated separately from the derivatization with 1-naphthylisothiocyanate, the two-fold ion-pair liquid–liquid-extraction and the treatment with hydrogen peroxide for the 2–20 ng/ml concentration range. The automatic procedure was preceded by a triple calcium precipitation and interrupted by evaporation of the SPE eluate under nitrogen. For the 0.5–5 μg/ml concentration range one automatic sequence was used by avoiding evaporation during the sample treatment. In addition to the labour-saving of the semi-automatic procedure, the daily sample-throughput was improved compared to the existing manual assay. Further, the validation showed marginal improvements in the precision, accuracy and lower limit of quantification.     

Quantitative determination of pamoic acid in dog and rat serum by automated ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography

Pamoic acid is used as a counter ion to obtain long-acting pharmaceutical formulations of certain basic drugs. In order to investigate the pharmacokinetics of pamoic acid, a simple, sensitive and reliable method has been established for the quantitative determination of pamoic acid in serum from dog and rat. The method uses ion-pair solid-phase extraction followed by ion-pair reversed-phase high-performance liquid chromatograpy. The influence on recovery of the addition of different agents (tetrabutylammonium acetate, methanol, sodium hydroxide) to the serum samples prior to solid-phase extraction was studied and the analytical method was validated. The method was found to be valid for accurate, precise and selective determination of pamoic acid in the tested concentration range of 5–200 ng/ml serum. The overall performance of the HPLC method was found to be satisfactory for the purpose of determining concentrations of pamoic acid in serum samples from pharmacokinetic studies with pamoic acid in dogs and rats.     

Simultaneous determination of nicotine and cotinine in plasma using capillary column gas chromatography with nitrogen-sensitive detection

A rapid and sensitive method is described for the simultaneous determination of nicotine and its principal metabolite, cotinine, in plasma. A one-step extraction procedure is employed and the quantitative analyses are performed by capillary column gas chromatography using a thermionic specific detector. Other special measures to avoid contamination from external sources such as atmosphere, solvents and laboratory equipment, which constitutes the major limiting factor of nicotine assay, were also undertaken. The structural analogues of nicotine and cotinine, N-methylanabasine and N-ethylnorcotinine, are used as internal standards. Moreover, a micromethod, which requires only 0.1 ml of plasma and found to be suitable for analysis of cotinine in finger-tip samples of blood, is described. Linearity over the concentration ranges 5–100 ng of nicotine per ml of plasma and 5–500 ng of cotinine per ml of plasma is demonstrated. The precision of the method has been investigated by determining the reproducibility at different levels of nicotine and cotinine within the working ranges, for both 1-ml and 0.1-ml samples of plasma.     

High-performance chromatographic assay for the sulphoxide metabolite of 2′-deoxy-3′-thiacytidine in human urine

A high-performance liquid chromatographic method is described for the determination in human urine of GI138870X, the sulphoxide metabolite of a novel dideoxynucleoside analogue, 2′-deoxy-3′-thiacytidine (lamivudine). GI138870X was extracted from human urine using Empore SDB RPS solid-phase extraction disks prior to reversed-phase chromatography with UV detection. The method has shown to be valid over the concentration range 0.5–100 μg/ml using a 0.5-ml sample volume.     

High-performance liquid chromatographic method for the quantitative determination of butorphanol, hydroxybutorphanol, and norbutorphanol in human urine using fluorescence detection

A sensitive, quantitative reversed-phase high-performance liquid chromatographic method has been established for the simultaneous determination of butorphanol, a synthetic opioid, and its metabolites, hydroxybutorphanol and norbutorphanol, in human urine samples. The method involved extraction of butorphanol, hydroxybutorphanol, and norbutorphanol from urine (1.0 ml), buffered with 0.1 ml of 1.0 M ammonium acetate (pH 6.0), onto 1-ml Cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the HPLC mobile phase, acetonitrile—methanol—water (20:10:70, v/v/v), containing 10 mM ammonium acetate and 10 mM TMAH (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5-μm column. The analysis was accomplished by detection of the fluorescence of the three analytes, at excitation and emission wavelengths of 200 nm and 325 nm, respectively. The retention times for hydroxybutorphanol, norbutorphanol, the internal standard, and butorphanol were 5.5, 9.0, 13.0, and 23.4 min respectively. The validated quantitation range of the method was 1–100 ng/ml for butorphanol and hydroxybutorphanol, and 2–200 ng/ml for norbutorphanol in urine. The observed recoveries for butorphanol, hydroxybutorphanol, and norbutorphanol were 93%, 72%, and 50%, respectively. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of study samples. The method was applied on study samples from a clinical study of butorphanol, providing a pharmacokinetic profiling of butorphanol.     

Determination of butorphanol in horse race urine by immunoassay and gas chromatography–mass spectrometry

An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC–MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC–MS system. The ELISA test (20 μl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC–MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC–MS method in the SIM mode using nalbuphine as internal standard included linearity studies (10–250 ng/ml), recovery (±100%), intra-assay (4.1–14.9%) and inter-assay (9.3–45.1%) precision, stability (10 days), limit of detection (10 ng/ml) and limit of quantitation (20 ng/ml).     

Urinary excretion of 19-norandrosterone of endogenous origin in man: quantitative analysis by gas chromatography–mass spectrometry

A GC–MS method, using deuterium-labelled 19-noretiocholanolone as internal standard and following an extensive LC purification prior to selected ion monitoring of the bis(trimethylsilyl) ethers at ion masses m/z 405, 419, 420 and 421, allowed the quantitation of subnanogram amounts of 19-norandrosterone present in 10-ml urine samples at m/z 405. Thirty healthy men, free of anabolic androgen supply, delivered 24-h urine collections in 4 timed fractions. Accuracy was proven by the equation, relating added (0.05–1 ng/ml) to measured analyte, which had a slope not significantly different from 1. Precision (RSD) was 4% at a concentration of 0.4 ng/ml, and 14% at 0.04 ng/ml. Analytical recovery was 82%. The limit of quantitation was 0.02 ng/ml. The excretion ranges were 0.03–0.25 μg/24 h or 0.01–0.32 ng/ml in nonfractionated 24-h urine.Taking into account inter-individual variability and log-normal distribution, a threshold of 19-norandrosterone endogenous concentration of 2 ng/ml, calculated as the geometric mean plus 4 SD, was established. This value corresponds to the decision limit advised by sport authorities for declaring positive (anabolic) doping with nandrolone.