Automated column-switching high-performance liquid chromatography for the determination of aflatoxin M1

An extractionless method for determining aflatoxin M1 (AFM1), a major metabolite of aflatoxin B1 (AFB1), in human urine was developed. The biological fluid is injected directly into the chromatographic system after simple dilution and centrifugation. A pre-column, packed with a cation-exchange phase and coupled on-line to a column-switching liquid chromatography (LC) system, is used for sample pre-treatment and concentration. The analytes are non-selectively desorbed with the LC eluent and cleaned by means of a column-switching procedure. Pre-treatment and analysis were performed within 40 min. Average AFM1 recovery reached 97% in the 10–100 ng/l range of urine. The detection limit of AFM1 in urine and milk was 2.5 ng/l for 1 ml of injected sample. A comparison with an immunoaffinity column clean-up and LC method was performed. The method was applied to determine AFM1 in the urine of AFB1 gavaged rats, and in the urine of both potentially exposed and supposedly unexposed workers. The method was also extended to milk.     

Trace determination of urinary 3-hydroxybenzo[a]pyrene by automated column-switching high-performance liquid chromatography

3-Hydroxybenzo[a]pyrene (3-OHB[a]P), one of the metabolites of benzo[a]pyrene (B[a]P), has been determined in human urine using an automated column-switching procedure. The hydrolysed biological sample is centrifuged just prior to being injected into a reusable precolumn loop, which is packed with a preparative phase and coupled on-line to a liquid chromatographic (LC) system. A rapid pre-treatment of the hydrolysed sample, consisting of a concentration and a crude clean-up, is performed on the precolumn. The analytes are then non-selectively desorbed with the LC eluent and the sample is cleaned again in three successive purification columns using the direct transfer or “heart-cut” technique. The pre-treatment does not exceed 3 min. and the entire analytical purification and separation procedure takes less than 30 min. Average 3-OHB[a]P recovery reaches 95% in the 1–50 ng/l range of urine, and the detection limit is 0.1 ng/l urine for a 3 ml injection of hydrolysed urine. The developed method was compared with a more time-consuming off-line method to analyse urines of B[a]P gavaged rats; the statistical treatment indicates that both methods are in agreement. The method was applied to purify and concentrate the urine samples of workers exposed and apparently unexposed to polycyclic aromatic hydrocarbons (PAHs).     

Fast and sensitive determination of urinary 1-hydroxypyrene by packed capillary column switching liquid chromatography coupled to micro-electrospray time-of-flight mass spectrometry

The present work reports capillary liquid chromatographic column switching methodology tailored for fast, sensitive and selective determination of 1-hydroxypyrene (1-OHP) in human urine using micro-electrospray ionization time-of-flight mass spectrometric detection. Samples (100 microl) of deconjugated, water diluted and filtered urine samples were loaded onto a 150 microm I.D.x 30 mm 10 microm Kromasil C(18) pre-column, providing on-line sample clean-up and analyte enrichment, prior to back flushed elution onto a 150 microm I.D.x 100 mm 3.5 microm Kromasil C(18) analytical column. Loading flow rates up to 100 microl/min in addition to the use of isocratic elution by a mobile phase composition of acetonitrile/water (70/30, v/v) containing 5 mM ammonium acetate provided elution of 1-OHP within 5.5 min and a total analysis time of less than 15 min with manual operation. Ionization was performed in the negative mode and 1-OHP was observed as [M-H](-) at m/z 217.08. The method was validated over the concentration range 0.2-40 ng/ml 1-OHP in pre-treated urine, yielding a coefficient of correlation of 0.997. The within-assay (n=6) and between-assay (n=6) precisions were in the range 6.4-7.3 and 7.0-8.1%, respectively, and the recoveries were in the range 96.2-97.5 within the investigated concentration range. The method mass limit of detection was 2 pg, corresponding to a 1-OHP concentration limit of detection of 20 pg/ml (0.09 nmol/l) diluted urine or 0.3 ng/ml (1.35 nmol/l) urine.     

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography–mass spectrometry

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography–mass spectrometry (GC–MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into a GC–MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 μg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.     

On-line solid-phase extraction and high-performance liquid chromatographic determination of chlorthalidone in urine

A simple and rapid on-line method for the determination of chlorthalidone in urine is proposed. The sample containing the internal standard is injected in a CN precolumn. After a 2-ml water rinsing, the precolumn is coupled for 30 s to the HPLC column via a switching valve, allowing the on-line elution of the compounds of interest. Analysis is carried out by reversed-phase chromatography with an acetonitrile-0.01 M phosphate buffer pH 7 (20:80, v/v) eluent, using UV detection at 214 nm. While the LC separation is performed, the precolumn is regenerated and conditioned, and is ready to receive the next sample at the end of the run. Accurate (>95%) and precise (<10%) analyses, in the range of 0.1–20 μg/ml of chlorthalidone in urine, have been achieved using this method.     

Determination of non-protein bound phenylbutazone in bovine plasma using ultrafiltration and liquid chromatography with ultraviolet detection

A liquid chromatographic procedure using UV detection was coupled with ultrafiltration for the quantitation of free phenylbutazone in bovine plasma, in the range of 20 ng/ml to 2.0 μg/ml. Whole plasma samples (0.5 to 1 ml) were placed in a 2-ml centrifugal concentrator with a molecular-mass cut-off membrane of 10 000 and centrifuged at 4500 g for 2 h at 4°C using a fixed angle rotor. The ultrafiltrate was transferred to an LC vial with a 200-μl insert and 100 μl was injected into an LC system. The chromatographic system used a C₁₈ reversed-phase column connected to a UV detector set at 264 nm. The mobile phase was 0.2 M sodium phosphate buffer (pH 7)–methanol (1:1). Recoveries of phenylbutazone from protein-free plasma water fortified at levels of 20 ng/ml to 2 μg/ml ranged from 91 to 93%, with relative standard deviations (R.S.D.s) ranging from 1 to 4%. The concentration of incurred non-protein bound phenylbutazone obtained from a cow intravenously dosed twice with 2 g phenylbutazone, 8 h apart, was 111, 26 and 11 ng/ml for 2, 72 and 104 h post first phenylbutazone dose, respectively.     

On-line solid-phase extraction of ceftazidime in serum and determination by high-performance liquid chromatography

A column-switching high-performance liquid chromatographic assay is described for the determination of ceftazidime (a third-generation cephalosporin) in human serum. The method does not require prior sample pretreatment. Serum is directly injected in a first chromatographic column for sample clean-up and extraction. Thereafter, using an on-line column-switching system, the drug is quantitatively transferred and separated on a second, analytical column followed by determination using ultraviolet absorption at 258 nm. The technique allows direct, rapid, precise, and simple determination of ceftazidime in serum over the range of 1–250 μg/ml using 12.5 μl of serum. This method was applied to study the pharmacokinetics of the drug in patients undergoing vascular surgery.     

Automated isolation of high-purity plasma albumin for isotope ratio measurements

Measurement of the incorporation of labeled amino acids in plasma albumin, isolated from plasma sampled at different time points after infusion start is a well-known technique to study human albumin synthesis. Unfortunately, no chromatographic method has been described yet, enabling the automated isolation of high-purity albumin from large numbers of plasma samples as is required to study the kinetics of this process. Therefore, we developed a fast protein liquid chromatographic method, capable of processing 200 μl amounts of plasma in 74 min (injection to injection). The system can run unattended as the FPLC system is connected to a sample processor equipped with a polyether ether ketone (PEEK) sample loop and a cooled sample tray. Albumin isolation was divided into three steps. First, plasma samples were injected onto a 1-ml Blue Sepharose HiTrap affinity column, equilibrated with 50 mmol/l phosphate buffer (pH 7.0). After elution of non-binding protein, switching the solvent to phosphate buffer with 1.5 mol/l sodium chloride eluted albumin. The resulting albumin fraction was desalted on-line by directing it through two consecutive HiTrap 5-ml desalting columns, whereafter it was retained in the system within a 5-ml PTFE loop, connected to a motor valve. After switching this valve, thus bypassing the sample loop, the phosphate buffers were changed automatically to Tris buffers. Final purification involved elution of the captured fraction over a 1-ml ion-exchange Resource Q column, using a sodium chloride gradient, ranging from 0 to 0.5 mol/l in Tris buffer (20 mmol/l, pH 7.5). A more than 99% purity of the final albumin fraction was confirmed by capillary electrophoresis.     

High-performance liquid chromatographic assay of l-α-glycerophosphorylcholine using a two-step enzymic conversion

A high-performance liquid chromatographic method using an enzymic reactor for determination of l-α-glycerophosphorylcholine in pharmaceutical forms is described. The procedure includes incubation of l-α-glycerophosphorylcholine with glycerophosphorylcholine phosphodiesterase (EC 3.1.4.2), giving choline and glycerophosphate, and subsequent chromatography of choline with a post-column enzymic reactor and electrochemical detection. The results obtained show a close linearity of the whole assay from 2 to 150 nmol/ml l-α-glycerophosphorylcholine, the sensitivity being 2 pmol per 20 μl of injected sample. The precision of the method in the analysis of l-α-glycerophosphorylcholine in pharmaceutical forms, ampoules and capsules, was 1.34 and 1.21%, respectively.     

Direct plasma liquid chromatographic-tandem mass spectrometric analysis of granisetron and its 7-hydroxy metabolite utilizing internal surface reversed-phase guard columns and automated column switching devices

An alternative on-line automated sample enrichment technique useful for the direct determination of various drugs and their metabolites in plasma is described for rapid development of highly sensitive and selective liquid chromatographic methods using mass spectrometric detection. The method involves direct injection of plasma onto an internal surface reversed-phase (ISRP) guard column, washing the proteins from the column to waste with aqueous acetonitrile, and backflushing the analytes onto a reversed-phase octyl silica column using switching valves. The analytes were detected using a tandem mass spectrometer operated in selected reaction monitoring (SRM) mode using atmospheric pressure chemical ionization (APCI). Use of two ISRP guard columns in parallel configuration allowed alternate injections of plasma samples on these columns for sample enrichment and shortened the column equilibration and LCMSMS analysis times, thereby increasing the sample throughput. The total run time, including both sample enrichment and chromatography, was about 6 min. Using this technique, an analytical method was developed for the quantitation of granisetron and its active 7-hydroxy metabolite in dog plasma. Granisetron is a selective 5-HT3 receptor antagonist used in the prevention and treatment of cytostatic induced nausea and vomiting. Recovery of the analytes was quantitative and the method displayed excellent linearity over the concentration ranges tested. Results from a three day validation study for both compounds demonstrated excellent precision (1.3–8.7%) and accuracy (93–105%) across the calibration range of 0.1 to 50 ng/ml using an 80 μl plasma sample. The automated method described here was simple, reliable and economical. This on-line approach using ISRP columns and column switching with LCMSMS is applicable for the quantification of other pharmaceuticals in pharmacokinetics studies in animals and humans which require high sensitivity.     

Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

Determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine using reversed-phase liquid chromatography with column switching and electrochemical detection

A column-switching method was developed for the determination of total 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in urine. This was performed by first treating samples with β-glucuronidase, followed by extraction with ethyl acetate. The reconstituted extracts with injected onto an HPLC system containing an amperometric detector and tandem Nucleosil C₁₈ and C₈ reversed-phase columns connected by a switching valve. The total analysis time for MHPG was 12 min. The limit of detection was 0.18 ng, or 9 μg/l for 20-μl injections of a 1.0-ml reconstituted extract prepared from 1.0 ml of urine. The linear range extended up to 80 mg/l. The within-day precision for a urine sample containing 170 μg/l total MHPG was ±6% and the day-to-day precision was ±15%. The average levels determined by this method for total MHPG in normal subjects showed good agreement with previous literature values. This approach could be modified for the determination of free MHPG by using only ethyl acetate extraction for sample pretreatment.     

Determination of methenamine in biological samples by gas—liquid chromatography

Methenamine (hexamethylenetetramine), a urinary disinfectant, was determined in human plasma and urine by gas—liquid chromatography with a short (10 m) open-bone glass capillary column (split ratio 1:20) and nitrogen-selective detector. An almost quantitative recovery (92.1%) was achieved by simple dilution of water-containing samples (0.5 ml) with acetone (4.5 ml). After centrifugation and aliquot (2 μl) of the supernatant was injected into the gas chromatograph. Selectivity and sensitivity of the nitrogen detector allowed the quantitation of unchanged methenamine in plasma and urine up to 24 h after a single therapeutic dose of 1 g.Reproducibility of the method was 7.6 and 2.1% (C.V.) in serum and urine, respectively. The time required for the analysis of one sample was approx. 2 min. Due to the simple extraction and short analysis time it was possible to analyze the samples concurrently with sample taking. Absorption of standard tablets and an enterosoluble preparation of methenamine hippurate was compared.     

High-performance liquid chromatography–tandem electrospray mass spectrometry for the determination of lidocaine and its metabolites in human plasma and urine

A sensitive, selective and accurate high-performance liquid chromatographic–tandem mass spectrometric assay was developed and validated for the determination of lidocaine and its metabolites 2,6-dimethylaniline (2,6-xylidine), monoethylglycinexylidide and glycinexylidide in human plasma and urine. A simple sample preparation technique was used for plasma samples. The plasma samples were ultrafiltered after acidification with phosphoric acid and the ultrafiltrate was directly injected into the LC system. For urine samples, solid-phase extraction discs (C₁₈) were used as sample preparation. The limit of quantification (LOQ) was improved by at least 10 times compared to the methods described in the literature. The LOQ was in the range 1.6–5 nmol/l for the studied compounds in plasma samples.     

High-performance liquid chromatographic determination of finasteride in human plasma using direct injection with column switching

A fully automated column-switching high-performance liquid chromatographic (HPLC) method was developed for the quantification of finasteride [N-(1,1-dimethylethyl)-3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide] in human plasma. Plasma samples were diluted with an equal volume of ethylene glycol-water (40:60, v/v), then the diluted sample (150 μl) was injected into the HPLC system without clean-up. The analyte was retained on a pretreatment column, whereas plasma proteins and other endogenous components were washed out to waste. The analyte was transferred to the analytical column in the heart-cut mode and then detected at 210 nm. A quantification limit of 1 ng/ml was attained. There was a linear relationship between peak height and drug concentration in plasma in the range 1–50 ng/ml. This method was validated and applied to the assay of plasma samples to characterize pharmacokinetic parameters in clinical studies.     

Determination of a chemoprotective agent, 2-(allylthio)pyrazine, in plasma, urine and tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a chemoprotective agent, 2-(allylthio)pyrazine (I), in human plasma and urine, and in rat blood and tissue homogenate using diazepam as an internal standard. The sample preparation was simple; 2.5 volumes of acetonitrile were added to the biological sample to deproteinize it. A 50–100 μl aliquot of the supernatant was injected onto a C₁₈ reversed-phase column. The mobile phase employed was acetonitrile–water (55:45, v/v), and it was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector at 330 nm. The retention times for I and the internal standard were 4.0 and 5.1 min, respectively. The detection limits of I in human plasma and urine, and in rat tissue homogenate (including blood) were 20, 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 6.1%) in a concentration range from 0.02 to 10 μg/ml for human plasma and urine, and for rat tissue homogenate. No interferences from endogenous substances were found.     

Determination of urinary thiocyanate and nitrate using fast ion-interaction chromatography

A simple and rapid chromatographic method for determination of nitrite, nitrate and thiocyanate is reported, and applied to the analysis of non-, medium and heavy smokers' urine samples. Ion-interaction liquid chromatography was carried out on a short 30 mm x 4.6 mm C18 column (3 microm particle size) with a mobile phase of 10 mM tetrabutylammonium phosphate in 20% MeOH. The chromatography was performed at an elevated temperature of 45 degrees C, at a flow-rate of 1 ml/min. Detection was by direct UV absorption at 230 nm. Sample preparation involved centrifugation and dilution, followed by sample clean-up on a C18 solid-phase extraction cartridge. The developed method proved both precise (% RSD <2%) and sensitive (standard detection limits <0.1 mg/l), and yielded total run times of under 10 min when applied to urine analysis of smokers and non-smokers, with thiocyanate eluting in under 5 min.     

Sensitive gas-liquid chromatographic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum or plasma

A gas-liquid chromatographic (GLC) assay suitable for the analysis of the cis(Z)-stereoisomer of the antipsychotic drug flupentixol in human serum or plasma was developed. The minimal quantifiable concentration was 0.5 ng/ml and the day-to-day coefficient of variation was 11.2% at 1 ng/ml and 8.7% at 10 ng/ml. Following addition of perphenazine as the internal standard (I.S.) and aqueous NaOH, samples (2 ml) are extracted with n-hexane-isoamyl alcohol (98.5:1.5, v/v) (solvent), back-extracted to 0.1 M HCl and after one washing-step and addition of aqueous NaOH again extracted into 100 μl solvent. After evaporation to dryness, the extract is reconstituted in 20 μl solvent and evaporated to approximative 10 μl. A 4-μl aliquot is injected cool on-column onto the GLC system. A gas chromatograph HP 5890 with on-column injection port, nitrogen-phosphorus detector (NPD), a HP-1 25 m × 0.32 mm I.D., 0.5 μm capillary and hydrogen (3 ml/min, automated pressure control) as the carrier gas was applied. The negative influence of light on the assay was measured and discussed. The suitability of this method for clinical pharmacokinetic studies and therapeutic drug monitoring (TDM) was determined by the analysis of serum samples of 12 schizophrenic patients.     

A novel clean-up method for urine analysis of low-molecular mass aldehydes by capillary electrophoresis with laser-induced fluorescence detection

A rapid clean-up method using ultra-filtration was developed to remove sample matrix in the determination of low-molecular mass aldehydes in human urine. The ensuing filtrate was derivatized with fluorescein 5-thiosemicarbazide and the labelled aldehydes determined by capillary zone electrophoresis with laser-induced fluorescence detection. Practical aspects related to the effect of the urine sample matrix on the label chemistry and the electrophoretic separation showed that the urine samples must be diluted 20-fold after their ultra-filtration. By using synthetic urine, linear ranges were established in the range of 15-5000 μg/l with limits of detection between 4.5 and 9 μg/l. The intra- and inter-assay precision (relative standard deviation, %) of the aldehydes ranged from 4.1% to 8.4% and 6.1%-9.6%, respectively, and the average specific uncertainty was 149±12 ng. The average recoveries performed on two levels by enriching synthetic urine samples ranged between 94% and 100%. Finally, the proposed method was applied to check low-molecular mass aldehydes in the human urine of a female volunteer to obtain information about the risk in her exposure to these chemicals in the workplace.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.