Liquid chromatographic determination of azidothymidine in human plasma using on-line dialysis and preconcentration on a silver(I)-thiol stationary phase

An automated method is described for the routine determination of 3′-azido-2′,3′-dideoxythymidine (AZT), the best known drug against acquired immunodeficiency syndrome (AIDS). The method is based on on-line dialysis to remove matrix macromolecules, followed by selective preconcentration and clean-up with a silver(I)-thiol stationary phase. After desorption of the solute with a small plug of perchloric acid, chromatography is applied using an octadecyl-modified silica column. Using UV absorbance detection at 269 nm, the minimum detectable concentration in plasma is 20 ng ml^?1 (600-μl sample). The within-day reproducibility at the 20 ng ml^?1 level is 4.4% and at least 128 samples can be analysed unattendedly without exchanging the dialysis membrane, the precolumn or the analytical column.     

Automated analysis of oxolinic acid and flumequine in salmon whole blood and plasma using dialysis combined with trace enrichment as on-line sample preparation for high-performance liquid chromatography

The use of dialysis as sample clean-up for high-performance liquid chromatography makes fully automated determination of drugs in whole blood and plasma possible. High recoveries of the analytes oxolinic acid and flumequine and the internal standard nalidixic acid are obtained after a short time of dialysis (7.3 min). The dilute dialysates are enriched on a small column packed with polystyrene. When dialysis is discontinued, the analytes are eluted by mobile phase to the analytical column. With UV detection the limit of detection was 50 ng/ml for both oxolinic acid and flumequine. Validation showed good precision and accuracy and good correlation between determinations in plasma and whole blood.     

The Analysis of Sulphonamide Drug Residues in Pork Muscle using Automated Dialysis

ABSTRACT

The aim of this study was to assess the suitability of automated dialysis, using a commercial system, for the analysis of sulphonamides in porcine tissue. The system involves automated dialysis, followed by trace enrichment of the dialysate prior to HPLC determination. The procedure was applied to the analysis of nine sulphonamide drug residues using reversed phase HPLC as the method of determination. Muscle samples were blended in saline, centrifuged and the supernatant was filtered before dialysis for an optimised time of 11 min. The resulting dialysate was concentrated on a reversed phase trace enrichment cartridge prior to HPLC analysis with UV detection at 280 nm. The developed method was evaluated by carrying out intra- and inter-assays on fortified porcine muscle. Mean recoveries, evaluated from the inter-assay study, were 80% or higher for the nine sulphonamides studied and the limit of determination for the method was 40 ng g^?1.     

Fully automated high-performance liquid chromatographic analysis of whole blood and plasma samples using on-line dialysis as sample preparation. Determination of oxytetracycline in bovine and salmon whole blood and plasma.

A fully automated technique for high-performance liquid chromatographic analysis of whole blood and plasma is described. Samples are automatically injected into a dialyser where proteins and blood cells are removed. The dialysates are concentrated on a small column prior to analysis. This technique is used for the determination of oxytetracycline in whole blood and plasma. After dialysis oxytetracycline and the internal standard, tetracycline, are retained on a polystyrene enrichment column and subsequently separated on a polystyrene analytical column by ion-pair chromatography. Using ultraviolet detection 50 ng/ml can be detected. Validation showed good within-day and between-day accuracy and precision. Different oxytetracycline concentrations were found in plasma and whole blood. This difference varied between the species.     

Monitoring of veterinary drug residues by a combination of continuous flow techniques and column-switching high-performance liquid chromatography. I. Sulphonamides in egg, meat and milk using post-column derivatization with dimethylaminobenzaldehyde

By using a combination of a low-pressure continuous flow module and a column-switching high-performance liquid chromatographic system, procedures have been developed for the automated residue analysis of sulphonamides and selected other drugs in meat, egg and milk. Aqueous extracts are purified by on-line dialysis and subsequent trace enrichment on a short column containing silica-based or polymer material. After backflush to the analytical column, detection is performed either at 280 nm or, after post-column derivatization with p-dimethylaminobenzaldehyde, at 450 nm. With these procedures the fully automated determination of both polar and apolar sulphonamides and dapsone above 5-20 micrograms/kg is possible. Coefficients of variation are 4-10%, recoveries compared to standards are 85-90%. The methods developed were tested in routine veterinary drug control.     

On-line dialysis and weak cation-exchange enrichment of dialysate. Automated high-performance liquid chromatography of pholcodine in human plasma and whole blood.

An automated method for the determination of pholcodine in plasma and whole blood is described. The technique combines dialysis and trace enrichment prior to high-performance liquid chromatography. Dialysis, trace enrichment on a weak cation-exchange column, separation on a cyano column and fluorescence detection was shown to be an extremely selective and sensitive method. The method has been used successfully in the analysis of real samples after administration of pholcodine. The automated method can be used, after minor modification, to determine other basic drugs in whole blood and plasma.     

High Performance Liquid Chromatographic Determination of Neomycin in Milk Using a Hisep Column

Abstract

A high performance liquid chromatographic (HPLC) method for the determination of neomycin in milk is described. Milk is passed directly through an amberlite CG-50 ion exchange resin column, and the neomycin which is retained on the column is derivatized with ortho-phthalaldehyde (OPA) reagent. The derivatized neomycin is eluted from the column with potassium borate buffer/methanol and analyzed by HPLC. A HISEP HPLC column with fluororoetric detection was used. Recoveries ranged from 94 to 102% in samples fortified between 0.1 to 5ppra levels. The detection limit is 50ppb.     

LC–MS/MS method for simultaneous determination of serum p‐cresyl sulfate and indoxyl sulfate in patients undergoing peritoneal dialysis

p‐Cresol sulfate (pCS) and indoxyl sulfate (IS) are protein‐bound uremic toxins that accumulate in patients with chronic kidney disease (CKD). They are closely associated with the mortality rate of CKD and morbidity of cardiovascular disease. In the present study, we established a rapid method for determination of pCS and IS by HPLC‐MS/MS in serum samples from 205 CKD patients undergoing peritoneal dialysis. In brief, serum was extracted by acetonitrile and spiked with hydrochlorothiazide. The prepared sample was eluted through HPLC column (Agilent Zorbax SB‐C₁₈, 3.5 μm, 2.1 × 100 mm) with a mobile phase of acetonitrile and 10 mm ammonium acetate solution (10:90, v/v) for subsequent detection of pCS and IS by MS/MS. The linearity ranged from 50 to 10,000 ng/mL for pCS (r > 0.99), and from 500 to 10,000 ng/mL for IS (r > 0.99). The lower limit of quantification was 50 ng/mL for pCS, and 500 ng/mL for IS. Relative standard deviation (RSD) of intra‐ and inter‐day precision was within ±15%. The results showed that pCS and IS levels were partially correlated with renal function in CKD patients, and IS was directly related to serum creatinine and estimated glomerular filtration rate.     

Stepwise injection photometric determination of nickel in air aerosols

A procedure is developed for the automated determination of nickel in air aerosols; it involves the adhesive separation of aerosols on a fiberglass column in the on-site mode followed by the photometric determination of analytes with dimethyl glyoxime under the conditions of stepwise injection analysis of aerosol concentrates. The analytical range for nickel is 1.5–38 μg/m³; the detection limit of the method is 0.5 μg/m³ at an air sample volume of 30 L. The duration of sampling to an adhesive column and concentrate analysis were 15 and 10 min, respectively.     

On-line dialysis-SPE-CE of acidic drugs in biological samples

A fully automated method is presented for the determination of acidic drugs in urine and serum using on-line dialysis-solid-phase extraction (SPE)-capillary electrophoresis (CE) with UV detection. With non-steroidal anti-inflammatory drugs (NSAIDs) as test compounds, detection limits in the biological samples were 0.05-1.0 microgram ml-1. Calibration plots were linear over two orders of magnitude and the within-day and between-day repeatability were better than 10%. The CE capillary and SPE column were used for over 500 analyses; the dialysis membrane was replaced after 250 analyses. A general protocol for dialysis-SPE-CE which can be used for amphoteric and acidic drugs was devised. The present results show that this protocol has general validity and can be recommended for future work on other classes of drugs.     

Determination of betaine,l‐carnitine,and choline in human urine using a self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A simple method for the determination of betaine, l ‐carnitine, and choline in human urine was developed based on column‐switching ion chromatography coupled with nonsuppressed conductivity detection by using a self‐packed column. A pretreatment column (50 mm × 4.6 mm, id) packed with poly(glycidyl methacrylate‐divinylbenzene) microspheres was used for the extraction and cleanup of analytes. Chromatographic separation was achieved within 10 min on a cationic exchange column (150 mm × 4.6 mm, id) using maleic anhydride modified poly(glycidyl methacrylate‐divinylbenzene) as the particles for packing. The detection was performed by ion chromatography with nonsuppressed conductivity detection. Parameters including column‐switching time, eluent type, flow rates of eluent, and interfering effects were optimized. Linearity (r ² ≥ 0.99) was obtained for the concentration range of 0.50–100, 0.75–100, and 0.25–100 μg/mL for betaine, l ‐carnitine, and choline, respectively. Detection limits were 0.12, 0.20, and 0.05 μg/mL for betaine, l ‐carnitine, and choline, respectively. The intra‐ and interday accuracy and precision for all quality controls were within ±10.11%. Satisfactory recovery was observed between 92.5 and 105.0%. The validated method was successfully applied for the determination of betaine, l ‐carnitine, and choline in urine samples from healthy people.     

Simultaneous Determination of Carbamazepine and its Active Metabolite Carbamazepine-10,11-epoxide in Human Plasma by UPLC

A simple method for the determination of carbamazepine and its active metabolite carbamazepine-10,11-epoxide by ultra performance liquid chromatography (UPLC) with ultraviolet absorbance detection (TUV) was developed. The method involves a two-step protein precipitation by liquid–liquid extraction. Phenytoin sodium was used as the internal standard. The separation was carried out on Acquity C18 column with acetonitrile:methanol:KH₂PO₄ buffer (adjusting pH to 4.6 with 85% o-phosphoric acid) (180/180/170, v/v/v) as the mobile phase at a flow rate of 0.4 mL min⁻¹. Linear detection response was obtained for concentrations ranging from 50 to 5,000 ng mL⁻¹. The limit of quantification (LOQ) was 50 ng mL⁻¹. The method was validated successfully for the determination of carbamazepine and its active metabolite carbamazepine-10,11-epoxide, which can be applied through pharmacokinetics and bioequivalence studies.

     

Simultaneous analysis of multiple urinary biomarkers for the evaluation of oxidative stress by automated online in‐tube solid‐phase microextraction coupled with negative/positive ion‐switching mode liquid chromatography–tandem mass spectrometry

This study described an automated online method for the simultaneous determination of 8‐isoprostane, 8‐hydroxy‐2′‐deoxyguanosine, and 3‐nitro‐l ‐tyrosine in human urine. The method involves in‐tube solid‐phase microextraction using a Carboxen 1006 PLOT capillary column as an extraction device, followed by liquid chromatography with tandem mass spectrometry using a CX column and detection in the negative/positive switching ion‐mode by multiple reaction monitoring. Using their stable isotope‐labeled internal standards, each of these oxidative stress biomarkers showed good linearity from 0.02 to 2.0 ng/mL. Their detection limits (S/N = 3) were 3.4–21.5 pg/mL, and their intra‐ and inter‐day precisions (relative standard deviations) were >3.9 and 6.5% (n = 5), respectively. This method was applied successfully to the analysis of urine samples, without any other pretreatment and interference peaks.     

Solid Phase Extraction of Lead(II) by Sorption on Grinded Eucalyptus Stem and Determination with Flame Atomic Absorption Spectrometry

A sensitive and simple solid‐phase preconcentration procedure for the determination of trace amount of lead by flame atomic absorption spectrometry (FAAS) is developed. The method is based on the adsorption of Pb²⁺ on the column of fine grinded eucalyptus stem adsorbent, elution of the column by nitric acid and subsequent determination by FAAS. The effect of different variables such as pH, eluent type, flow rate and interfering ions on the recovery of the analyte was investigated and optimum conditions were established. The adsorption of lead onto fine grinded eucalyptus stem can formally be described by a Langmuir equation with a maximum adsorption capacity of 4.49 mg g^?1. A preconcentration factor of 50 was achieved using the optimum conditions. The calibration graph was linear in the range 10–125 ng mL^?1 of lead in the initial solution with r = 0.9982. The limit of detection based on 3S_b criterion was 4.5 ng mL^?1 and the relative standard deviation for eight replicate measurements of 30 and 80 ng mL^?1 of iron was 3.6 and 2.8%, respectively. The method was successfully applied to the determination of lead added to well, tap and wastewater samples.     

Determination of butoxyacetic acid in urine by capillary gas chromatography

Summary A simple, rapid and reliable method is presented for the determination of butoxyacetic acid (BAA), the main metabolite of ethylene glycol monobutyl ether (Butyl Cellosolve). The urine is acidified with hydrochloric acid and passed through a cation-exchange column. BAA which is quantitatively found in the filtrate is subsequently adsorbed on XAD-4 resin. After desorption with diethyl ether an aliquot of the eluate is evaporated in a nitrogen stream and methylated with diazomethane in diethyl ether. A forty-fold enrichment is achieved by this procedure. The gas-chromatographic separation is performed on a 60 m fused silica capillary column DB-1 (100% dimethyl-polysiloxane). Flame ionization is used for detection. Pentoxyacetic acid (PAA) serves as internal standard. The detection limit of BAA in urine is 0.02 mg/l. Linearity has been tested up to 50 mg/l. The losses by the clean-up steps are between 10.0% and 22.7%. The average recovery is 100.9%. Within-series imprecision (n=10) has been determined for three concentrations and ranges between 4.8% and 12.6%.

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Capillar-gas-chromatographische Bestimmung von Butoxyessigsäure in Harn

     

High Performance Liquid Chromatogrphy of Fat-Soluble Vitamins. III. Determination of Vitamin D3 in Pharmaceutical Preparations and in Blood

Abstract

A reversed phase high-performance liquid chromatographic method (HPLC) is described for separation and determination of colecalciferol (Vitamin D₃) in Vitamin preparations and in biological materials. Vitamin D₃ is extracted from the formulations and from the blood in a fully automated electronically controlled extraction apparatus. For HPLC a column of lichrosorb RP18 and methanol as eluent are used. The extraction, separation and determination of vitamin D₃ needs about 10–20 minutes. The described extraction and HPLC methods allow the detection of 1–2 ng per injection and are well reproduced with a maximum coefficient of variation of < 3,5%. Vitamin A-acetate is used as internal standard.     

Trace Determination of Domoic Acid in Sea Water and Phytoplankton by High-Performance Liquid Chromatography of the Fluorenylmethoxycarbonyl (FMOC) Derivative

Abstract

A method is presented for the trace determination of domoic acid, a neurotoxic amino acid responsible for cases of Amnesic Shellfish Poisoning resulting from the consumption of contaminated shellfish. The method involves pre-column derivatization with 9-fluorenylmethylchloroformate to form the FMOC derivative followed by reversed-phase HPLC with fluorescence detection. The detection limit for domoic acid in seawater and aqueous extracts is 15pg/mL (50 pM) using gradient elution, a 20μL injection volume, and a 2.1mm I.D. microbore column. Use of dihydrokainic acid as an internal standard improved quantitation. The method was applied to the detection of domoic acid in seawater, in phytoplankton cultures (Nitzschia pungens forma multiseries), and in natural mixed phytoplankton assemblages in estuarine waters.     

Determination of phenols in waters using micro-pumped multicommutation and spectrophotometric detection: an automated alternative to the standard procedure

An automated and greener spectrophotometric procedure has been developed for the determination of phenol in water at 700 nm. The method uses the reaction between phenol, sodium nitroprusside, and hydroxylamine hydrochloride in a buffered medium at pH 12.3. The flow manifold comprises four solenoid micro-pumps employed for sample and reagent introduction into the reaction coil and to transport the colored product formed to the detector. The linear dynamic range was 50–3,500 ng mL⁻¹ (R = 0.99997; n = 6) and the method provided a limit of detection (3σ) of 13 ng mL⁻¹. The sampling throughput was estimated to be 65 measurements per hour and the coefficient of variation was 0.5% (n = 10) for a 1.0 μg mL⁻¹ phenol concentration. Recoveries of 92–105% were obtained for phenol determination in spiked water samples at concentration levels from 50 to 5,000 ng mL⁻¹. The use of multicommutation reduced the reagent consumption 25-fold, the sample consumption 225-fold, and the waste generation 30-fold compared with the batch procedure. The proposed method is an environmentally friendly alternative to the official 4-aminoantipyrine method since it avoids the use of chloroform.     

Optimisation of a dialytic set-up for liquid chromatography: automated separation and preconcentration of ciprofloxacin

Continuous-flow and static dialysis coupled on-line to liquid chromatography was evaluated and an automated method for determination of ciprofloxacin in biological samples developed. A trace enrichment column packed with C18 material and coupled with a continuous dialysis and reversed-phase HPLC system with fluorescence detection enabled determination of ciprofloxacin in human blood serum at the 0.1-nmol/l level. The amount of analyte preconcentrated and loaded on the HPLC system was linearly proportional to the concentration in the dialysed sample over more than 4 orders of magnitude (up to 1 x 10(-6) M). Data for linearity, repeatability and detectability for each particular set-up are given. The trace enrichment step eliminates band broadening caused by solvents different from those of the eluent and affecting retention of ciprofloxacin on the analytical column (increase in k') due to the on-column change of eluent composition. In analysis of human serum samples phthalates leached from plastic materials may interfere due to coelution with the analyte.     

Liquid chromatographic determination of fluvastatin and its enantiomers in blood plasma by automated solid-phase extraction

Summary Liquid chromatographic methods for determination of fluvastatin, as racemate and asseparated enantiomers, are described. Fluvastatin is an anticholesterolemic agent that inhibits the rate limiting enzyme in cholesterol biosynthesis. The sample clean-up was fully automated, using solid-phase extraction. Plasma samples were injected onto disposable C2 cartridges, connected on-line with the analytical column. After washing, the analytes were eluted and transferred to a C8-analytical column for racemate determination or to a Chiralcel OD-R column for enantiomer separation. Analytes were monitored by fluorescence detection after post-column exposure of the eluate to UV light, which enhanced the sensitivity 20-fold for fluvastatin and 10-fold for the enantiomers. Absolute recoveries were >90% both for the recemate and enantiomers. Limits of quantitation were 0.5 nM for the racemente and 1 nM for enantiomers, using 200 μL plasma sample. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996