Simultaneous high-performance liquid chromatographic determination of salicylates in whole blood, plasma and isolated erythrocytes

A method using liquid—liquid extraction has been developed for the isolation of acetylsalicylic acid and its metabolites, salicylic, gentisic or possibly salicyluric acids, from whole blood, isolated erythrocytes and plasma. Methylene chloride proved to be the best of the organic solvents tested. For whole blood and isolated erythrocytes it was necessary to carry out haemolysis prior to their extraction. The high-performance liquid chromatographic conditions for the quantitation of acetylsalicylic acid and its metabolites from samples of whole blood, erythrocytes and whole plasma were optimized. Separation was performed using reversed-phase chromatography on Separon SGX C₁₈ and ultraviolet detection at 236 nm. A mixture of methanol—water (80:100, v/v) was the mobile phase, acidified with perchloric acid to pH 2.5.     

High-performance liquid chromatographic assay for amiloride in plasma and urine

A sensitive and simplified high-performance liquid chromatographic procedure has been developed for quantification of amiloride in rabbit plasma, as well as human plasma and urine. Following protein precipitation with perchloric acid, the supernatant was directly injected into a C₁₈ Nucleosil column. The mobile phase consisted of methanol—water (45:55) containing 0.1 M perchloric acid, and the compound was quantitated using a fluorescence detector at excitation and emission wavelengths of 286 and 418 nm, respectively. The average recovery was 97.6%. The calibration curve was linear over the range 2.0–20.0 ng/ml. The limit of detection was 0.5 ng/ml.     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C₁₈ solid-phase extraction column. After elution with 500 μl of elution solution, the penicillins are derivatized with 500 μl of 1,2,4-triazole—mercuric chloride solution at 65°C for 30 min. The penicillin—mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C₁₈ column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

Determination of isofezolac in biological fluids by reversed-phase liquid column chromatography

A rapid, sensitive, and specific reversed-phase high-performance liquid chromatography assay was developed for the determination of 1,3,4-triphenylpyrazole-5-acetic acid (isofezolac) in plasma and urine. The assay involves extraction into diethyl ether from plasma buffered at pH 4.4. The organic phase is evaporated and the residue, dissolved in the mobile phase [acetonitrile—water—0.2 M phosphate buffer (pH 3) (65 : 15 : 20)] is chromatographed at a flow-rate of 1.5 ml/min. The drug is detected by its UV absorption (detection limit 100 ng/ml) or its very intense fluorescence (detection limit 10 ng/ml). Absolute analytical recoveries for isofezolac varied from 92.9 to 100.4%. The accuracy is ca. 1%. Each separation requires about 6 min. This method was applied successfully to the determination of isofezolac in humans for pharmacokinetic studies.     

Determination of very-long-chain fatty acids in plasma by a simplified gas chromatographic—mass spectrometric procedure

The concentration of very-long-chain fatty acids (VLCFA) (straight chain, more than 22 carbon atoms) in plasma or in cultured fibroblasts is one of the most important diagnostic criteria for the diagnosis of the peroxisomal disorders. A sensitive method for VLCFA assay in plasma, using small sample volume and a simplified procedure, is described. After adequate extraction and derivatization, methyl esters of VLCFA are separated, identificated and quantified by gas chromatography—mass spectrometry (GC—MS). The method is sensitive, reproducible, accurate and relatively simple. GC—MS equipment used for routine organic acid analysis can be used.     

Determination of plasma fatty acid composition in neonates by gas chromatography

Total fatty acids in plasma of neonates have been analysed as their methyl esters by gas chromatography. They were separated on a capillary column coated with a SP-2380 stationary phase. As little as 100 μl of plasma is used for the analysis. The extraction procedure was performed with dichloromethane—methanol (2:1) and fatty acids were methylated with boron trifluoride—methanol. The quantification of fatty acids is based on an internal standard method. Absolute values (μg fatty acid per 100 μl plasma) are given together with relative values (%). At a signal-to-noise ratio of 3, the detection limits for flame ionisation detection are between 0.08 to 0.51 ng. The high sensitivity and precision permits the effective determination of the fatty acids in neonate plasma.     

Gas chromatographic—mass fragmentographic determination of homopantothenic acid in plasma

A gas chromatographic—mass fragmentographic method was developed for the determination of homopantothenic acid in plasma. Acidified plasma was deproteinized by extraction with chloroform and subsequently the aqueous layer was extracted with ethyl acetate. The organic layer containing homopantothenic acid was reduced to dryness, and the resulting residue was redissolved in N,O-bis(trimethylsilyl)trifluoroacetamide—pyridine solution to allow trimethylsilylation. Aliquots of this solution were injected into the gas chromatograph—mass spectrometer and analyzed by the selected ion monitoring method using l-ascorbic acid as an internal standard. The detection limit for homopantothenic acid was 5 ng/ml of plasma.A precise and sensitive assay for the determination of homopantothenic acid in plasma was established.     

Measurement of catecholamines in biological fluids by high-performance liquid chromatography : A comparison of fluorimetric with electrochemical detection

An improved method for the determination of catecholamines in biological fluids, by reversed-phase high-performance liquid chromatography (HPLC) with fluorimetric detection is presented. The pH titration previously employed in the alumina extraction was abandoned in favour of the use of a molar excess of pH 8.5 Tris—HCl buffer. A novel lyophilisation step serves to concentrate the catechols and by reconstituting in mobile phase, chromatography disturbances are minimised. The addition of 2 mM octanesulphonic acid to a citrate—phosphate mobile phase at pH 6.0 gave optimal resolution and sensitivity.That HPLC separation can improve the specificity of the trihydroxyindole reaction, to the extent of providing a reliable analytical method, has been demonstrated and validated by the technique of HPLC with electrochemical detection. A correlation coefficient of 0.98 was obtained between the two techniques as applied to the measurement of urinary catecholamines. The HPLC—fluorimetric method was sensitive enough to measure 0.1 ng/ml of noradrenaline or adrenaline at a signal-to-noise ratio of 2.0. Application of the method to the quantitative determination of catecholamines in human urine, plasma and rat brain homogenates is demonstrated.     

Determination of glibenclamide in human plasma by solid-phase extraction and high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma has been developed. Sample clean-up prior to chromatographic analysis was accomplished by extraction of the drug using a solid-phase RP-8 or RP-18 cartridge instead of the conventional liquid-liquid extraction methods described. For the separation of the drug from the endogenous components a reversed-phase column (LiChrosorb RP-8) of 5 μm particle size and 250×4 mm I.D., together with a mobile phase consisting of acetonitrile-12 μM perchloric acid (47:53) was selected. The method employs progesterone as an internal standard, and a reversed-phase column combined with UV detection of the drug at 230 nm. The detector response was linear up to the concentration of 400 ng/ml and the average recovery was 100.36%. The sensitivity of the method was 5 ng/ml.     

Determination of picogram levels of heptylphysostigmine in human plasma using high-performance liquid chromatography with fluorescence detection

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid—liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50–2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.     

A sensitive method for determining quinolinic acid in animal tissues

A sensitive chromatographic method for isolation and measurement of quinolinic acid from rat liver and kidney is described. The method is based on the isolation of quinolinic acid by ion-exchange chromatography. The extraction of quinolinic acid consisted of the freeze clamping of the organ in liquid nitrogen, followed by deproteinization in perchloric acid. The neutralized extract was concentrated by freeze-drying and submitted to the action of concentrated perchloric acid to hydrolyze the nucleotides which interfered in the chromatographic separation of quinolinic acid. The sample was applied to a column of Dowex (HCOO^?) and eluted with a linear gradient of formic acid. The eluted fraction containing quinolinic acid was quantitatively measured by its absorbance at pH 2 and 268 nm in a spectrophotometer.     

Determination of timiperone in rat plasma by high-performance liquid chromatography with electrochemical detection

We report a sensitive new method for the determination of timiperone in rat plasma by using high-performance liquid chromatography with electrochemical detection. The method involves extraction of plasma samples with heptane-isoamyl alcohol at pH>8, followed by back-extraction into dilute acetic acid. Separation was accomplished by reversed-phase high-performance liquid chromatography on an ODS column with the mobile phase consisting of 0.1 M phosphate buffer (pH 3.5)-acetonitrile-methanol (65:20:15, v/v). Recovery was greater than 80%. Calibration curve was linear over the concentration range 0.5–50.0 ng/ml. The limit of quantitation of timiperone was 0.5 ng/ml plasma.     

Determination of diclofenac in plasma using a fully automated analytical system combining liquid—solid extraction with liquid chromatography

A fully automated analytical system based on liquid—solid extraction combined with column liquid chromatography is described for the determination of diclofenac in plasma. After addition of pH 5 buffer and the internal standard solution to the plasma sample, both sample preparation via a C₁₈ disposable extraction column and injection were performed by a Gilson ASPEC system. Diclofenac and the internal standard were separated on a reversed-phase column, using methanol—pH 7.2 phosphate buffer (56:44, v/v) as mobile phase at a flow-rate of 0.4 ml/min. The reproducibility and accuracy of the method were acceptable over the concentration range 31–3140 nmol/l in plasma.     

Simultaneous determination of catecholamines and dobutamine in human plasma and urine by high-performance liquid chromatography with fluorimetric detection

We report a reliable fluorimetric assay for the simultaneous determination of norepinephrine, epinephrine, dopamine and dobutamine in human plasma and urine, based on liquid—liquid extraction and derivatization with the fluorogenic agent 1,2-diphenylethylenediamine prior to chromatography. The method is sensitive (detection limit 0.3–0.8 pg injected) and reproducible (coefficients of variation 1–10%), and shows good accuracy (93–98%). The method should also be used when one only wants to measure the concentrations of the natural catecholamines, in order to avoid interference by metabolites of dobutamine and by the late-eluting dobutamine itself.     

High-performance liquid chromatographic determination of ambroxol in human plasma

Ambroxol has been determined in biological fluids using a rapid and sensitive high-performance liquid chromatographic method. The samples prepared from plasma by liquid—liquid extraction were analysed on reversed-phase silica gel by competing-ion chromatography with ultraviolet detection. The method was applied to the determination of ambroxol levels in twelve healthy volunteers after oral administration of 90 mg of ambroxol in tablets of Mucosolvan and Ambrosan.     

Determination of carbimide in plasma by gas—liquid chromatography

A sensitive and selective method for the measurement of carbimide, the hydrolytic product of calcium carbimide, in plasma is described. The procedure involves extraction with ethyl acetate, derivatization with heptafluorobutyric anhydride and analysis by gas—liquid chromatography with electron-capture detection. The lower limit of sensitivity of the assay is 5.0 ng/ml carbimide in plasma. The overall accuracy of the procedure is 96.1% with a coefficient of variation not exceeding 8.7%. This assay has been used to investigate the time-course of plasma carbimide concentration in the rat following oral administration of calcium carbimide.     

Determination of the aza alkyl lysophospholipid 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine in rat plasma by liquid chromatography—particle beam—mass spectrometry

A sensitive and specific method for the determination of the aza alkyl lysophospholipid (AALP) 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine (I) in rat plasma is described. The target molecule was analyzed by high-performance liquid chromatography (HPLC)—mass spectrometry (MS) after one single liquid—liquid extraction with chloroform—methanol (2:1, v/v). 1,2-Didecanoyl-sn-glycero-3-phosphocholine was used as internal standard. HPLC was carried out using a polymeric reversed-phase column; the coupling to the mass spectrometer was a particle beam (PB) interface, and the ionization method was electron impact (EI). This simple and rugged method permits the measurement of I in rat plasma in the range of 25 ng/ml–5 μg/ml with good accuracy and precision and is used in pharmacokinetic studies.     

Method for separation and determination of lactone and hydroxy acid forms of a new HMG CoA reductase inhibitor (RG 12561) in plasma

The new drug RG 12561 (I) is a lactone that is undergoing clinical evaluation for its cholesterol lowering effect based on potent HMG CoA reductase inhibitory activity displayed by its open hydroxy acid form. To determine the dispositional characteristics of the drug, a method was developed for determination of the two forms in plasma. A 0.25-ml aliquot of plasma was deproteinized with 0.5 ml of methanol, and the lactone was extracted with hexane—ethyl acetate (75:25, v/v). The methanolic plasma was then acidified followed by extraction of the hydroxy acid with hexane—ethyl acetate. The extracts were dried, reconstituted and analyzed by isocratic, reversed-phase high-performance liquid chromatography using ultraviolet absorbance at 254 nm. The separations were performed utilizing a C₁₈ column with mobile phase consisting of acetonitrile, 2-propanol and 0.1 M acetate buffer (pH 5), the proportions of which differed depending on the form of drug analyzed. The method was found to be selective and a quantitation limit of 50 ng/ml was established. Validation studies demonstrated that the method was sufficiently accurate and precise for determining disposition of the drug in the dog.     

Simplified method for simultaneous determination of diazepam and its metabolites in urine by thin-layer chromatography and direct densitometry

A direct densitometric method for determination of diazepam and its metabolites in urine was developed. The proposed procedure involves acid hydrolysis of urine specimens, thereby converting diazepam and its metabolites into benzophenones [2-methylamino-5-chlorobenzophenone (MACB) and 2-amino-5-chlorobenzophenone (ACB)]. It is followed by extraction with chloroform—isopropanol (3:1, v/v). The two benzophenones were separated on thin-layer chromatography plates using hexane—diethyl ether—acetic acid (80:10:10) as a mobile phase. Quantitation of the MACB and ACB spots was achieved by direct ultraviolet densitometry. The limit of detection was 0.5 μg per ml of urine for both benzophenones. The proposed method is simple, rapid, reproducible and has been found to be effective for direct determination of diazepam and its metabolites in urine.     

Simultaneous Determination of Fluzinamide and three of its active metabolites in plasma by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method has been developed for a new anticonvulsant, fluzinamide, and three of its active metabolites. This method requires only 0.5 ml of plasma, and it involves a single extraction with a mixture of hexane—dichloromethane—butanol (55:40:5). The plasma extract is chromatographed on a 10-μm, C₁₈ reversed-phase column and quantitated by ultraviolet absorbance at 220 nm. The concentration—response curve for all four compounds are linear from 0.05 μg/ml to at least 10 μg/ml. The extraction efficiency of this method is greater than 90%. The accuracy and precision of the method were tested by analyzing spiked unknown samples that had been randomly distributed across the concentration range. The mean concentrations found were within ± 9% of the various amounts added with a standard deviation of ± 3.5%. This method has been successfully applied to the analysis of samples obtained from fluzinamide-dosed dogs, healthy unmedicated volunteers, and patients who were at steady state with phenytoin, carbamazepine, and fluzinamide.