Improved selectivity of a high-performance liquid chromatography assay for debrisoquine and its 4-hydroxy metabolite from urine

Debrisoquine is an antihypertensive drug that displays polymorphic metabolism and has been used to determine hydroxylator status in human subjects. A high-performance liquid chromatographic method for the simultaneous urine analysis of debrisoquine (D) and its primary metabolite, 4-hydroxydebrisoquine (4-OHD), is described. A cyanopropyl (CN) extraction column and CN analytical column were used with a mobile phase consisting of 20% acetonitrile/20 mM sodium dihydrogen orthophosphate (pH = 7.1) at a flow rate of 1.5 ml/min. The peaks of interest, as well as the internal standard, were eluted from the column in less than 14 min. The method described was validated for within-day and between-day accuracy and precision for both D and 4-OHD. Coefficients of variation at all concentrations tested were less than 4.1% for D and less than 9.3% for 4-OHD. The assay was used to evaluate D/4-OHD ratios, thereby determining hydroxylation status in 65 healthy male volunteers.     

Determination of paracetamol in urine by liquid chromatography

Since the biotransformation of paracetamol (Acetaminophen) is practically confined to conjugation, the quantitative determination of paracetamol excretion may provide important information on phase II of the drug metabolism. We elaborated a simple and rapid liquid chromatographic method for the assessment of paracetamol and its conjugated metabolites in the urine to be available for routine use in the clinicopharmacological laboratory. The persons involved in the trial were administrated 500 mg of paracetamol to be taken on an empty stomach in the morning. Subsequently, their urine was collected for 8 hours. The so-called free paracetamol of unchanged form excreted into the urine was measured from this 0 to 8 hours' urine fraction, then, after treating it with beta-glucuronidase/arysulphatase enzyme, the total amount of paracetamol released from the conjugate, as well as that of the existing free paracetamol, the so-called total paracetamol were determined. The urine extracts containing paracetamol obtained by ethylacetate, at pH 10, and dried under nitrogen stream were analysed by HPLC on an ODS column in an eluent of methanol and water mixture (3:7, v/v) in the presence of 3-acetaminophenol internal standard. The flow rate was 1 ml/min, the detection wavelength was 254 nm.     

3,4-Dehydrodebrisoquine, a novel debrisoquine metabolite formed from 4-hydroxydebrisoquine that affects the CYP2D6 metabolic ratio.

Considerable unexplained intersubject variability in the debrisoquine metabolic ratio (urinary debrisoquine/4-hydroxydebrisoquine) exists within individual CYP2D6 genotypes. We speculated that debrisoquine was converted to as yet undisclosed metabolites. Thirteen healthy young volunteers, nine CYP2D6*1 homozygotes [extensive metabolizers (EMs)] and four CYP2D6*4 homozygotes [poor metabolizers (PMs)] took 12.8 mg of debrisoquine hemisulfate by mouth and collected 0- to 8- and 8- to 24-h urines, which were analyzed by gas chromatography-mass spectrometry (GCMS) before and after treatment with beta-glucuronidase. Authentic 3,4-dehydrodebrisoquine was synthesized and characterized by GCMS, liquid chromatography-tandem mass spectrometry, and (1)H NMR. 3,4-Dehydrodebrisoquine is a novel metabolite of debrisoquine excreted variably in 0- to 24-h urine, both in EMs (3.1-27.6% of dose) and PMs (0-2.1% of dose). This metabolite is produced from 4-hydroxydebrisoquine in vitro by human and rat liver microsomes. A previously unstudied CYP2D6*1 homozygote was administered 10.2 mg of 4-hydroxydebrisoquine orally and also excreted 3,4-dehydrodebrisoquine. EMs excreted 6-hydroxydebrisoquine (0-4.8%) and 8-hydroxydebrisoquine (0-1.3%), but these phenolic metabolites were not detected in PM urine. Debrisoquine and 4-hydroxydebrisoquine glucuronides were excreted in a highly genotype-dependent manner. A microsomal activity that probably does not involve cytochrome P450 participates in the further metabolism of 4-hydroxydebrisoquine, which we speculate may also lead to the formation of 1- and 3-hydroxydebrisoquine and their ring-opened products. In conclusion, this study suggests that the traditional metabolic ratio is not a true measure of the debrisoquine 4-hydroxylation capacity of an individual and thus may, in part, explain the wide intragenotype variation in metabolic ratio.     

Rapid determination of atenolol in human plasma and urine by high-pressure liquid chromatography

A rapid, specific, high-pressure liquid chromatographic determination of atenolol in plasma and urine was developed. This method employs the high sensitivity of fluorescence detection together with selective extraction and reversed-phase chromatography to measure concentrations as low as 20 ng of drug/ml of plasma with a coefficient of variation of 3.91%. The assay is specific enough to be valid in the presence of plasma and urine substances. The detection limit (i.e., three times baseline noise) is 3 ng/ml.     

Determination of linezolid in human serum and urine by high-performance liquid chromatography.

An HPLC method is described for the determination of the new oxazolidinone antibiotic linezolid (I) in human biofluids. After precipitation of serum proteins with perchloric acid the protein free supernatant was separated by isocratic reversed-phase chromatography on a Nucleosil-100 5C18 column. The mobile phase consisted of a mixture of acetonitrile: sodium acetate buffer: water (180:100:720, v/v) adjusted to pH 3.7. Urine was diluted with aqueous buffer solution. The column eluate was monitored at 250 nm. Validation of the method yielded satisfactory results for serum (and urine); detection limit 0.07 mg/l (2.4), lower limit of quantitation 0.14 mg/l (4.7), linear range 20 mg/l (500), imprecision within series (c.v.) 1.8-2.5% (0.8-1.0), imprecision between series (c.v.) 1.8-9.3 (0.4-9.3), recovery 99-102% (93-103). Comparison of HPLC results with results obtained using a quantitative microbiological assay yielded acceptable agreement both for serum and urine. The method was successfully used in a pharmacokinetic study with human volunteers.     

Micromethod for determination of ceftriaxone in plasma and urine by high-performance liquid chromatography

A sensitive and rapid high-performance liquid chromatographic method for the determination of ceftriaxone in human plasma and urine is described. A C18 reversed phase column is used; the mobile phase comprises water-methanol-triethylamine (750:250:4v/v/v) adjusted to pH 3 with orthophosphoric acid. Quantitation is performed at 270 nm with cefazolin as the internal standard. This method involves precipitation of proteins from fluids with acetonitrile followed by extraction of endogenous compounds with chloroform and injection of the upper aqueous phase on to the chromatograph. Relative standard deviations for between-day and within-day assays are 6.2%. The detection limit is 0.5 mug(-1) in plasma and urine. Studies of drug stability during sample storage, sample pretreatment and chromatography showed no degradation of ceftriaxone or of the internal standard. The method is convenient for clinical monitoring and for pharmacokinetic studies.     

全自动二维高效液相色谱测定霉酚酸血药浓度的研究

目的：采用全自动二维液相色谱（2D-LC-UV）建立测定霉酚酸（MPA）血药浓度的方法,并应用于临床。方法：样品去除蛋白后直接进样,待测物在一维柱AstonSC2T （3.5 mm×50 mm,5 μm）上初步分离,通过中间柱Aston SBR（3.0 mm×10 mm,5 μm）截取保留,转移到二维柱Aston SBX4（4.6 mm×150 mm,5 μm）上进一步分离。一维流动相为MPA-1A移动相,流速0.8 mL·min^-1;二维流动相为MPA-2A移动相,流速1.2 mL·min^-1;柱温40℃;紫外检测波长304 nm。结果：在所建立的色谱条件下,霉酚酸与各杂质分离良好,在0.32~25.03 μg·mL^-1范围内与峰面积呈良好的线性关系（r=0.999 9）,可在13 min内完整出峰,方法回收率高于95%;日内、日间的精密度RSD值均小于5%;稳定性试验RSD小于5%。结论：本方法简便快速、结果准确、稳定性良好、自动化程度高、检测成本低,适用于临床霉酚酸快速检测。     

Determination of procaine in equine plasma and urine by high-performance liquid chromatography.

The variability in plasma and urine equine procaine measurement between three independent laboratories using current methods led to the development of a sensitive, reliable, and reproducible high-performance liquid chromatographic method. Standardbred mares were administered either a penicillin G procaine preparation intramuscularly or procaine hydrochloride subcutaneously, and blood and urine were collected at defined time intervals. By HPLC the detection limits for procaine in plasma and urine were 1 and 10 ng/mL, respectively. In contrast procaine in plasma could not be detected by GC-NPD, while the urinary detection limit was 50 ng/mL. The concentration of fluoride in the collection tubes and repetitive freeze-thawing modified plasma procaine measurement. Urinary pH was a factor in estimation of urine procaine levels with greater recovery and reproducibility of results at pH 5 as compared to pH 7. This HPLC method provides a simple, sensitive, and reliable quantitation of procaine in equine plasma and urine.     

Determination of theophylline and its metabolites in human urine by high-performance liquid chromatography

High-performance liquid chromatographic method with UV detection was developed for the determination of theophylline and its metabolites, in human urine using β-hydroxyethyl theophylline (β-HET) as an internal standard. For extraction of urine sample, quality control sample and xanthine-free blank urine were mixed with decylamine (ion-paring reagent) and β-HET. After saturation with ammonium sulfate, the mixture was then extracted with organic solvent at pH values of 4.0∼4.5. All separations were performed with ion-pair chromatography using decylamine as an ion-pairing reagent and 3 mM sodium acetate buffered mobile phase (pH 4.0) containing 1% (v/v) acetonitrile and 0.75 mM decylamine. The detection limits of theophylline, 1,3-DMU, 1-MU, 3-MX and 1-MX in human urine were 0.17, 0.17, 0.39, 0.19 and 0.19 μg/ml, based on a signal-to-noise ratios of 3.0. The mean intraday coefficients of variation (C.V.s) of each compound on nine replicates were lower than 2.0%, while mean interday C.V.s on three days were lower than 1.6%. All separations were finished within 40 minutes.     

Simultaneous determination of cefepime and grepafloxacin in human urine by high-performance liquid chromatography

A liquid chromatographic method with UV detection for simultaneous determination of cefepime and grepafloxacin has been developed. The method uses a C18 column, equipped with a pre-column of the same material, and acetonitrile-0.1 M phosphoric acid/sodium hydroxide buffer (pH 3.0)-0.01 M n-octylamine (pH 3.0) as mobile phase in gradient mode. Mobile flow rate and sample volume injected were 1.3 mL min(-1) and 20 microL, respectively. Detection wavelengths were 259 nm for cefepime and 278 nm for grepafloxacin. The retention times were 4.03 min for cefepime and 8.85 min for grepafloxacin, with detection limits of 1.0 and 1.1 microg mL(-1), respectively. The method was applied to the determination of both antibiotics in spiked samples of human urine.     

Measurement of aflatoxin and aflatoxin metabolites in urine by liquid chromatography-tandem mass spectrometry

Automated immunoaffinity solid-phase extraction followed by liquid chromatography-tandem mass spectrometry and chemical analogue internal standardization is employed to detect and quantify the aflatoxins AFB(1), AFB(2), AFG(1), AFG(2), and the metabolites AFM(1) and AFP(1) in urine. The dynamic range of the method is nearly three orders of magnitude with limits of detection in the low femtogram on column range. The method was validated over a 12-day period by eight analysts. This method is suitable for agricultural, forensic, and public health laboratories during an accidental outbreak or a chemical terrorism event where a rapid and accurate diagnosis of aflatoxicosis is needed.     

Determination of barbiturates in urine by micellar liquid chromatography and direct injection of sample

A liquid chromatographic procedure for the determination of six barbiturates (barbital, diallyl barbituric acid, phenobarbital, butabarbital, amobarbital and pentobarbital) in urine samples is described. The proposed system uses a Spherisorb octadecyl-silane ODS-2 C18 analytical column and a guard column of similar characteristics. The UV detector was set at 240 nm. A study to select adequate composition of the micellar mobile phase for the separation of these compounds in urine samples is performed. Maximum resolution was achieved with a 0.07 M sodium dodecylsulphate-0.3% propanol at pH 7.4 eluent. Limits of detection at 240 nm were ranged between 0.13 microg ml(-1) for diallyl barbituric acid and 2.7 microg ml(-1) for amobarbital. The procedure allows for the determination of these compounds in 20 minutes, it does not require prior a sample preparation step and it can be very useful to the investigation of intoxication.     

Determination of erythromycin in serum and urine by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic analysis of erythromycin in human serum and urine with UV detection at 200 nm is presented. The method involves a solid-phase extraction procedure followed by a simple phase separation step and chromatography on a reversed-phase column. The method has sensitivity limits of 0.25 and 1.0 micrograms/mL in serum and urine, respectively, and is sufficiently sensitive to monitor concentrations of erythromycin in human serum and urine after the administration of a single 500-mg erythromycin stearate tablet.