Simple high-performance liquid chromatographic method for the determination of tolbutamide and its metabolites in human plasma and urine using photodiode-array detection

A high-performance liquid chromatographic method has been developed for the determination of tolbutamide and its metabolites in human plasma and urine. The compounds examined were extracted with diethyl ether from the acidified biological fluid. Chlorpropamide was used as internal standard, and 235 nm was chosen as the wavelength for diode-array detection. A study of the relationship between the capacity factor and the mobile phase composition and pH showed that acetonitrile-2-propanol-0.1% orthophosphoric acid (17: 17: 66, v/v) was the best eluent on a C8 reversed-phase column. The method is precise, sensitive and suitable for pharmacological investigations.     

Simultaneous determination of metacavir and its metabolites in rat plasma using high-performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS)

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the simultaneous determination of metacavir and its two metabolites in rat plasma was developed and validated. Tinidazole was used as an internal standard and plasma samples were pretreated with one‐step liquid–liquid extraction. In addition, these analytes were separated using an isocratic mobile phase on a reverse‐phase C₁₈ column and analyzed by MS in the selected reaction monitoring mode. The monitored precursor to product‐ion transitions for metacavir, 2′,3′‐dideoxyguanosine, O‐methylguanine and the internal standard were m/z 266.0 → 166.0, m/z 252.0 → 152.0, m/z 166.0 → 149.0 and m/z 248.0 → 202.0, respectively. The standard curves were found to be linear in the range of 1–1000 ng/mL for metacavir, 5–5000 ng/mL for 2′,3′‐dideoxyguanosine and 1–1000 ng/mL for O‐methylguanine in rat plasma. The precision and accuracy for both within‐ and between‐batch determination of all analytes ranged from 2.83 to 9.19% and from 95.86 to 111.27%, respectively. No significant matrix effect was observed. This developed method was successfully applied to an in vivo pharmacokinetic study after a single intravenous dose of 20 mg/kg metacavir in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification and determination of phase II nabumetone metabolites by high-performance liquid chromatography with photodiode array and mass spectrometric detection

Chromatographic analyses play an important role in the identification and determination of phase I and phase II drug metabolites. While the chemical standards of phase I metabolites are usually available from commercial sources or by various synthetic, degradation or isolation methods, the phase II drug metabolites have usually more complicated structures, their standards are in general inaccessible and their identification and determination require a comprehensive analytical approach involving the use of xenobiochemical methods and the employment of hyphenated analytical techniques. In this work, various high-performance liquid chromatography (HPLC) methods were employed in the evaluation of xenobiochemical experiments leading to the identification and determination of phase II nabumetone metabolites. Optimal conditions for the quantitative enzymatic deconjugation of phase II metabolites were found for the samples of minipig bile, small intestine contents and urine. Comparative HPLC analyses of the samples of above-mentioned biomatrices and of the same biomatrices after their enzymatic treatment using beta-glucuronidase and arylsulfatase afforded the qualitative and quantitative information about phase II nabumetone metabolites. Hereby, three principal phase II nabumetone metabolites (ether glucuronides) were discovered in minipig's body fluids and their structures were confirmed using liquid chromatography (LC)-electrospray ionization mass spectrometric (MS) analyses.     

Improved high-performance liquid chromatographic determination of ciprofloxacin and its metabolites in human specimens.

A simple approach to the quantitation of ciprofloxacin and its three metabolites, M1 (desethylene-ciprofloxacin), M2 (sulfo-ciprofloxacin) and M3 (oxo-ciprofloxacin), in human serum, urine, saliva and sputum is described. This assay allows the parent drug and its metabolites to elute and be resolved in a single chromatogram at 280 nm using a linear gradient. The procedure involved liquid-liquid extraction. Separation was achieved on a C18 reversed-phase column. The limit of detection of ciprofloxacin is 0.05 microgram/ml and that of its three metabolites is 0.25 microgram/ml. This method is sufficiently sensitive for pharmacokinetic studies.     

Extraction, quantitative gas chromatographic determination and gas chromatographic/mass spectrometric detection of ecgonine for identification of cocaine and its metabolites in urine

Summary Quantitative extraction studies from urine were carried out by addition of cocaine, benzoylecgonine, ecgonine methyl ester and ecgonine to urine samples. After hydrolysis to ecgonine the compounds were analyzed together. Ecgonine was isolated by a cation-exchange resin and purified by an anion-exchange resin. The quantitative determination was performed by GC after silylation with MSTFA. The recovery was 77% at a concentration of 150 g ecgonine/ml urine. A qualitative determination of ecgonine by GC/MS was possible up to the detection limit of 20 ng/ ml. The method can be applied for the detection of cocaine abuse.

---

Extraktion, quantitative gas-chromatographische Bestimmung und gas-chromatographischer massenspektrometrischer Nachweis von Ecgonin zur Identifizierung von Cocain und seinen Metaboliten in Urin

Zusammenfassung Es wurden quantitative Untersuchungen zur Extraktion von Urin durchgeführt, dem Cocain, Benzoylecgonin, Ecgoninmethylester und Ecgonin zugesetzt wurden. Die Summe dieser Verbindungen kann über eine vorgeschaltete Hydrolyse zu Ecgonin indirekt bestimmt werden. Nach Hydrolyse wird Ecgonin an einem Kationenaustauscher isoliert und der Extrakt an einem Anionenaustauscher gereinigt. Die quantitative Bestimmung erfolgt gaschromatographisch nach Silylierung mit MSTFA. Dabei beträgt die Wiederfindungsrate 77% bei Konzentrationen von 150 g Ecgonin/ml Urin. Der qualitative Nachweis von Ecgonin durch GC/MS ist bis zu einer Nachweisgrenze von 20 ng/ml möglich. Damit eignet sich die Methode zum Nachweis einer Cocain-Einnahme im Urin.

---

---

From the dissertation by U. Zerell, University of Bonn, 1986 (in preparation)     

Achiral and chiral determination of tramadol and its metabolites in urine by capillary electrophoresis

An achiral and chiral separation for the determination of tramadol and its main metabolite O-demethyltramadol in urine samples by CE with UV detection was developed. It was possible to separate tramadol and its phase I and phase II metabolites in one single run using a borate buffer. Furthermore, the simultaneous chiral separation of tramadol and the phase I metabolites was achieved using carboxymethyl-beta-cyclodextrin as chiral selector. To reach the required limits of quantification for the analytes, a preconcentration by solid-phase extraction for the achiral assay and by liquid-liquid extraction for the chiral assay was used. The methods were validated and their applicability was shown by the determination of tramadol and O-demethyltramadol in urine samples.     

Extraction of maleic hydrazide residues from potato crisps and their determination using high-performance liquid chromatography with UV and atmospheric pressure chemical ionisation mass spectrometric detection

A method was required for the determination of maleic hydrazide residues in potato crisps. A published method for the extraction of the analyte from onions and potatoes was evaluated and found to be inappropriate due to the inability of the extracting solvent to penetrate the oily matrix. A method was developed to overcome this problem; the resulting recovery data (mean=92.9%. R.S.D.=8.3%, N=16) confirmed its efficiency, and was used to analyse 48 retail potato crisp samples. To confirm possible residues identified by screening with HPLC-UV, and HPLC-atmospheric pressure chemical ionization MS method was developed. There was good agreement between the data obtained from the detection techniques (R²=0.978, slope=1.11).     

Rapid high-performance liquid chromatographic method for the measurement of atenolol in plasma using UV detection

A rapid, selective and reproducible high-performance liquid chromatographic method has been developed for the measurement of the beta-adrenoceptor blocking drug atenolol in small (400 microliters) volumes of plasma. Following solid phase sample preparation using Bond-ElutTM mini-columns the compound is separated by high-performance column liquid chromatography on a microparticulate (6 microns) cyano column using acetonitrile--ammonium dihydrogen phosphate (4:96) containing triethylamine (0.25%, v/v) as the mobile phase, and the absorption of the column effluent is monitored at 224 nm. The practical limit of quantitation, based upon an assay volume of 400 microliters, is 25 ng/ml for atenolol. The average coefficient of variation is 3.1%.     

Mass spectrometric and high-performance liquid chromatographic studies of medroxyprogesterone acetate metabolites in human plasma.

Medroxyprogesterone acetate (MPA) treatment has been shown to exert several beneficial effects in cancer patients. It has been suggested that such effects are due in part to the metabolites derived from MPA in vivo. The first results are reported on the identification of 2 alpha-hydroxy- and 21-hydroxy-MPA, 20-dihydro-MPA, 17 alpha-acetoxy-2 alpha,3 beta-dihydroxy-6 alpha-methylpregn-1,4-dien-20-one and two X,21-dihydroxy-MPAs, one of them presumably being 6 alpha-hydroxymethyl-21-hydroxy-MPA, in patient's plasma by high-performance liquid chromatographic (HPLC), gas chromatographic-mass spectrometric and NMR methods. Additionally, the presence of other metabolites such as di- and tetrahydro-MPAs and 6,21-dihydroxy-MPA, found in urine and other samples, was demonstrated in plasma. For routine clinical examinations an HPLC method is described for determination of, e.g., the unreduced MPA metabolite group in Sep-Pak-ODS column extracts of patients' plasma.     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection.

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene-divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.     

Sensitive liquid chromatographic/tandem mass spectrometric method for the determination of beclomethasone dipropionate and its metabolites in equine plasma and urine

Beclomethasone dipropionate (BDP) is a potent pro-drug to beclomethasone (BOH) and is used in the treatment of chronic and acute respiratory disorders in the horse. The therapeutic dose of BDP (325 microg per horse) by inhalation results in very low plasma and urinary concentrations of BDP and its metabolites that pose a challenge to detection and confirmation by equine forensic laboratories. To solve this problem, a method involving the use of a liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the detection, confirmation and quantification of the analytes in equine samples. Ammonium formate or acetate buffer added to LC mobile phase favored the formation of [M + H](+) ions from BDP and its metabolites, whereas formic acid led to the formation of sodium and potassium adduct ions ([M + Na](+), [M + K](+)) together with [M + H](+) ions. Acetonitrile, on the other hand, favored the formation of abundant solvent adduct ions [M + H + CH(3)CN](+) with the analytes under electrospray ionization (ESI) and atmospheric pressure chemical ionization conditions. In contrast, methanol formed much less solvent adduct ions than acetonitrile. The solvent adduct ions were thermally stable and could not be completely desolvated under the experimental conditions, but they were very fragile to collision-induced dissociation (CID). Interestingly, these solvent adduct ions were observed on a triple-quadrupole mass spectrometry but not on an ion trap instrument where helium used as a damping gas in the ion trap might cause the solvent adduct ions desolvated by collision. By CID studies on the [M + H](+) ions of BDP and its metabolites, their fragmentation paths were proposed. In equine plasma at ambient temperature over 2 h, BDP and B21P were hydrolyzed in part to B17P and BOH, respectively, but B17P was not hydrolyzed. Sodium fluoride added to equine plasma inhibited the hydrolysis of BDP and B21P. The matrix effect in ESI was evaluated in equine plasma and urine samples. The method involved the extraction of BDP and its metabolites from equine plasma and urine samples by methyl tert-butyl ether, resolution on a C(8) column with a mobile phase gradient consisting of methanol and ammonium formate (2 mmol l(-1), pH 3.4) and multiple reaction monitoring for the analytes on a triple-quadrupole mass spectrometer. The detection limit was 13 pg ml(-1) for BDP and B17P, 25 pg ml(-1) for BOH and 50 pg ml(-1) for B21P in plasma and 25 pg ml(-1) for BOH in urine. The method was successfully applied to the analysis of equine plasma and urine samples for the analytes following administration of BDP to horses by inhalation. B17P, the major and active metabolite of BDP, was detected and quantified in equine plasma up to 4 h post-administration by inhalation of a very low therapeutic dose (325 microg per horse) of BDP.     

Improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its N-demethylated metabolites in plasma using solid-phase extraction

An improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its N-demethylated metabolites in plasma is described. Excellent resolution of all components is provided by reversed-phase chromatography using a mobile phase consisting of 1% acetic acid-methanol (83:17) at a flow-rate of 2.7 ml/min, in conjunction with a Waters Assoc. Nova-Pak C18 column which was protected by a Waters Assoc. Guard-Pak precolumn module containing a Guard-Pak CN cartridge. Rapid extraction of caffeine and the dimethylxanthines from plasma was achieved using reversed-phase octadecylsilane bonded-silica columns (Bond-Elut C18). With only 100 microliters of sample, plasma levels in the region of 50 ng/ml for the dimethylxanthines and 100 ng/ml for caffeine can be determined using ultraviolet detection at 273 nm. The method has been used for measuring umbilical cord plasma samples to provide information regarding foetal exposure to caffeine and its metabolites and is also suitable for therapeutic drug monitoring of caffeine and theophylline levels in the treatment of neonatal apnoea.     

Case of enantiomer impurity identification by normal-phase chiral high-performance liquid chromatography with optical rotation and mass spectrometric detection

An impurity produced in the synthesis of compound I is separated and identified as its enantiomer II using normal-phase chiral high-performance liquid chromatography (HPLC) with UV absorbance, optical rotation (OR) and mass spectrometric (MS) detection. The results show that the impurity II and compound I have equal and opposite specific rotations, identical MS spectra and the same MS-MS fragmentation pattern, as required for enantiomers. The procedures presented demonstrate a novel combination of methods for enantiomer identification and characterization that do not require the preparation of individual enantiomer markers or even the racemic mixture, thus reducing the need for additional synthetic work.     

Thermospray high-performance liquid chromatography/mass spectrometric determination of cyclosporins.

The cyclic undecapeptides cyclosporin (Csp) A, CspD and dihydro CspC (HCspC) were analyzed by high-performance liquid chromatography/thermospray-mass spectrometry (HPLC/TSP-MS) on line with a UV detector. Positive ion partial (1000-1300 u) mass spectra of these compounds could be obtained with 1-2 pmol injected on column. Mass spectra were characterized by signals corresponding to the [M+H] ions as well as fragment ions derived from the loss of 112 (CspA and CspD) or 44, 114 and 114 + 44 (HCspC) mass units from the parent ion. The same qualitative profile was observed for negative-ion acquisition where the ions were formed by proton abstraction. The application of the technique to the characterization of CspA and its major hydroxylated and dealkylated metabolites in human blood samples is presented.     

Determination of nalbuphine using high-performance liquid chromatography coupled to photodiode-array detection and gas chromatography coupled to mass spectrometry.

A procedure involving capillary column gas chromatography coupled to mass spectrometry and a method involving liquid chromatography coupled to a diode-array detector have been developed for the analysis of nalbuphine. The extraction step is the same for both techniques and involves extraction under alkaline conditions in chloroform-2-propanol-n-heptane (50:17:33, v/v/v) with levallorphan as the internal standard. After purification by acidic extraction and back alkaline extraction, drugs are derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane for gas chromatography-mass spectrometry and directly injected for high-performance liquid chromatography-diode-array detection. The limits of detection are 2.0 and 25.0 ng/mg, respectively.     

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C(18) column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2x10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N=3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain.     

Ultra-performance liquid chromatography/tandem mass spectrometric determination of testosterone and its metabolites in in vitro samples

This paper describes the development and partial validation of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of testosterone (T) and its four metabolites, 6beta-OH-T, 16alpha-OH-T, 16beta-OH-T and 2alpha-OH-T, in in vitro samples. The analytical method involves direct dilution of samples with acetonitrile containing an internal standard, followed by separation of testosterone and the four metabolites on an Acquity UPLCtrade mark C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using turbo ionspray ionization. The parent compound and its metabolites investigated were well separated (Rs >1.5) with a run time of 4 min under a gradient condition. The method was partially validated. The linear concentration range was 0.01 to 5 microM for all the compounds of interest. Inter-assay mean bias and relative standard deviation (RSD) were in the range of -12% to 8% and 4.1% to 8.5%, respectively. Intra-assay mean bias and RSD were in the range of -8.0% to 5.2% and 3.4% to 9.6%, respectively. The lower limit of quantitation for this assay was 0.01 microM. The differences in LC/MS performance were investigated by conducting a comparison of UPLC with another method previously optimized for HPLC-based separation and quantification of testosterone and its metabolites.     

Liquid chromatographic/mass spectrometric determination of desmedipham and phenmedipham and their metabolites in soil

A simple method is described for the simultaneous determination of residues of 2 carbamate herbicides (phenmedipham and desmedipham) and related metabolites (m-aminophenol, aniline, and m-toluidine) in soil. The analytes are extracted from spiked soils with methanol. The solvent/soil suspension is centrifuged, and the supernatant is directly injected, without any further cleanup, into a reversed-phase liquid chromatography/mass spectrometry apparatus equipped with a TurbolonSpray interface. The method was tested on 5 soils having different physicochemical properties. Recoveries from the soil types, spiked over the range of 50-200 ppb, were essentially quantitative for each analyte. The detection limits of the method are < or = 25 ng/g.     

Activated alpha-alkyl-alpha-arylacetic acid enantiomers for stereoselective thin-layer chromatographic and high-performance liquid chromatographic determination of chiral amines

The separation of racemic benoxaprofen into the two benoxaprofen enantiomers by preparative high-performance liquid chromatography and the application of the activated enantiomers as derivatization reagents for the simultaneous stereoselective determination of chiral amines in biological material is described. Activated (+)- and (-)-benoxaprofen are both shown to be very sensitive and stable chiral fluorescence markers, applicable to thin-layer chromatography as well as to high-performance liquid chromatography.