Novel high-performance liquid chromatographic and solid-phase extraction methods for quantitating methadone and its metabolite in spiked human urine

A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from spiked human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200 μl) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrix were greater than 91% with RSD values less than 4.5%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP₁₈ column with a mobile phase of 0.1% TFA–methanol (60:40, v/v). Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signal-to-noise enrichment was achieved by diluting the final SPE eluates four-fold with water.     

Screening and determination of β-blockers, narcotic analgesics and stimulants in urine by high-performance liquid chromatography with column switching

A fast method is described for the screening of eleven β-blockers, two narcotic analgesics and two stimulants in urine by HPLC with column switching. The urine sample (100 μl), buffered tto pH 9–9.5, is injected onto a short extraction column packed with CN stationary phase. The extraction is flushed with water for 2.5 min to elute polar matrix components to waste. The retained components are then backflushed by means of a six-port valve onto the ODS analytical column where they are separated. Phosphate buffer pH 3.0 and acetonitrile were used as mobile phase. Gradient elution was applied in the screening method to improve separation. Detection was performed with diode-array detector at 220, 235 and 300 nm. Recoveries were near 100%, precision was excellent and sensitivity about 0.25 μg/1. The speed up the quantitative analysis, the same method but with isocratic elution was successfully applied to the determination of acebutolol and metoprolol in urine samples collected 4 h after administration of the compounds as single doses.     

High-performance liquid chromatographic determination of N-methylformamide and N-methyl-N-(hydroxymethyl)-formamide in human urine

A reliable high-performance liquid chromatographic (HPLC) method which allows the determination in human urine of two important metabolites of N,N-dimethylformamide (DMF), namely N-methylformamide (MMF) and N-methyl-N-(hydroxymethyl)formamide (DMFOH), is reported. A single-step rapid purification of urine was performed on a C₁₈ solid-phase extraction column and the eluate was injected directly on to the HPLC column. HPLC was carried out isocratically on Aminex Ion Exclusion HPX-87H column using 7.5 · 10⁻⁴ M sulphuric acid as the mobile phase with ultraviolet detection at 196 nm. The method is specific, accurate, precise and sufficiently sensitive to be applied to the biological monitoring of MMF and DMFOH in workers exposed to DMF.     

Column-switching high-performance liquid chromatographic assay for determination of apigenin and acacetin in human urine with ultraviolet absorbance detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of apigenin and the 4′-methylated derivative acacetin in human urine using column-switching and ultraviolet (UV) absorbance detection. Urine samples were enzymatically hydrolysed and solid-phase extracted prior to injection onto the HPLC system. Prior to elution of apigenin and the internal standard, 5,7,8-trihydroxyflavone, from the first column used for sample clean-up, the six-port valve was switched to the second column for analysis with UV detection. Detection of apigenin was precise and reproducible, with a limit of quantification of 10 ng ml⁻¹ urine. Detection and quantification of acacetin was linear down to 70 ng ml⁻¹ urine. The method has been successfully applied to determine the level of apigenin in 100 human urine samples from an intervention study with parsley.     

Extraction and separation of urinary catecholamines as their diphenyl boronate complexes using C18 solid-phase extraction sorbent and high-performance liquid chromatography

The clinical utility of a one-step extraction procedure based on the retention of a diphenyl boronate-catecholamine complex on a C18 solid-phase extraction sorbent was investigated for the measurement of urinary catecholamines. Although recoveries with the extraction procedure were optimal over a relatively broad pH range (7.5-9.5), analytical factors such as sample loading and elution flow-rates, wash step and elution conditions, the concentration of catecholamines in urine to be extracted and the type of C18 sorbent used for extraction were found to influence the efficiency of this procedure and would therefore need to be controlled for optimal recoveries. Under optimal conditions the recovery of noradrenaline, adrenaline and dopamine from spiked urine was high and reproducible (mean recoveries were >85% for all catecholamines). The effectiveness of sample clean-up step was demonstrated by reverse phase, ion pair high-performance liquid chromatography with electrochemical detection. The method described was found to be suitable for the routine measurement of catecholamines in urine in clinical biochemistry laboratories. It has a high sample extraction throughput (40/h) and has adequate precision (between batch CV<8%) and sensitivity (LOD<30 nmol/l; LOQ<65 nmol/l) for all the catecholamines measured. The method has acceptable accuracy, showing a mean bias of 6.6% for noradrenaline, 7.3% for adrenaline and 6.8% for dopamine from the mean value of laboratories (N=69) participating in an External Quality Assurance scheme for greater than 12 months.     

Simultaneous analysis of several non-steroidal anti-inflammatory drugs in human urine by high-performance liquid chromatography with normal solid-phase extraction

A practical and reproducible high-performance liquid chromatographic method using normal solid-phase extraction has been developed for the simultaneous analysis of twelve non-steroidal anti-inflammatory drugs (NSAIDs) in human urine. A urine specimen mixed with acetate buffer pH 5.0 was purified by solid-phase extraction on a Sep-Pak Silica cartridge. The analyte was chromatographed by a reversed-phase Inertsil ODS-2 column using a phosphate buffer-acetonitrile at pH 5.0 as the mobile phase, and the effluent from the column was monitored at 230 or 320 nm. Absolute recoveries were greater than 73% for all of the twelve NSAIDs. The present method enabled simple manipulation and isocratic HPLC with UV analysis as well as high sensivity of 0.005 μg/ml for naproxen, and 0.05 μg/ml for sulindac, piroxicam, loxoprofen, ketoprofen, felbinac, fenbufen, flurbiprofen, diclofenac, ibuprofen and mefanamic acid as the quantitation limit in human urine using indomethacin as an internal standard.     

High-performance liquid chromatographic analysis of 2',3'-dideoxyinosine in biological samples

A high-performance liquid chromatographic analysis for the anti-AIDS drug 2',3'-dideoxyinosine (ddI) in rat plasma and urine, with a limit of detection of 0.2 μg/ml and requiring a sample size of 100 μl is described. Diluted plasma or urine samples were extracted using a C₁₈ solid-phase extraction column. Retention of ddI on more polar solid-phase extraction columns was insufficient for sample clean-up. This method is useful for pharmacokinetic studies of ddI in small rodents.     

Determination of nalmefene in plasma by high-performance liquid chromatography with electrochemical detection and its application in pharmacokinetic studies

A method for measuring human plasma levels of nalmefene after oral and intravenous administration is presented. The method consists of a solid-phase extraction procedure followed by HPLC analysis. A cyanopropyl column is used for the solid-phase extraction and 60% (v/v) acetonitrile in dilute sodium pentanesulfonic acid solution for elution. The concentrated and filtered eluate is injected into the HPLC system, which is equipped with an electrochemical dual-electrode detector. A phenyl column is used in this HPLC system with a mobile phase containing 30% (v/v) acetonitrile in dilute sodium pentanesulfonic acid solution. A signal-to-noise ratio of 4.5 is obtained when a 1 ng/ml spiked plasma sample is analyzed. To determine the applicability of this method for human pharmacokinetic studies, nalmefene levels in plasma were measured at time points up to 24 h following oral and intravenous administration of 30 mg of nalmefene hydrochloride to two subjects. These studies demonstrated that the proposed method is sufficiently sensitive to study the pharmacokinetic profile of nalmefene in man.     

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 antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissue extracts using anion-exchange high-performance liquid chromatography and capillary gel electrophoresis

Chemically modified phosphorothioate oligodeoxynucleotides (ODNs) have become critical tools for research in the fields of gene expression and experimental therapeutics. Bioanalytical assays were developed that utilized fast anion-exchange high-performance liquid chromatography (HPLC) and capillary gel electrophoresis (CGE) for the determination of 20-mer ODNs in biological fluids (plasma and urine) and tissues. A 20 mer ODN in the antisense orientation directed against DNA methyltransferase (denoted as MT-AS) was studied as the model ODN. The anion-exchange HPLC method employed a short column packed with non-porous polymer support and a ternary gradient elution with 2 M lithium bromide containing 30% formamide. Analysis of the MT-AS is accomplished within 5 min with a detection limit of approximately 3 ng on-column at 267 nm. For plasma and urine, samples were diluted with Nonidet P-40 in 0.9% NaCl and directly injected onto the column, resulting in 100% recovery. For tissue homogenates, a protein kinase K digestion and phenol–chloroform extraction were used, with an average recovery of about 50%. Since the HPLC assay cannot provide one-base separation, biological samples were also processed by an anion-exchange solid-phase extraction and a CGE method to characterize MT-AS and its catabolites of 15–20-mer, species most relevant to biological activity. One base separation, under an electric field of 400 V/cm at room temperature, was achieved for a mixture of 15–20-mer with about 50 pg injected. Assay validation studies revealed that the combined HPLC–CGE methods are accurate, reproducible and specific for the determination of MT-AS and its catabolites in biological fluids and tissue homogenates, and can be used for the pharmacokinetic characterization of MT-AS.     

Amphetamine and methamphetamine determination in urine by reversed-phase high-performance liquid chromatography with sodium 1,2-napthoquinone 4-sulfonate as derivatizing agent and solid-phase extraction for sample clean-up

A rapid method is described for the identification and determination of amphetamine and methamphetamine in human urine samples by liquid chromatography with UV-Vis detection. The samples were transferred onto a C₁₈ solid-phase extraction column and chromatographed on a Hypersil ODS RP C₁₈, 5 μm (250 × 4 mm I.D.) with an acetonitrile-water elution gradient containing propylamine. Under these conditions, the amines are eluted with a short retention time. The procedure has been applied to the determination of amphetamine and methamphetamine in the range 0.3–4.0 μg/ml in spiked urine samples. The detection limits at 280 nm were 4 and 2 ng/ml for amphetamine and methamphetamine, respectively. The intra-day and inter-day precision and accuracy of the method were studied.     

Determination of Aconitum alkaloids in blood and urine samples. I. High-performance liquid chromatographic separation, solid-phase extraction and mass spectrometric confirmation

Determination of four toxic Aconitum alkaloids, aconitine, mesaconitine, hypaconitine and jesaconitine, in blood and urine samples has been established using high-performance liquid chromatography (HPLC) combined with ultraviolet absorbance detection, solid-phase extraction and mass spectrometry (MS). These alkaloids were hydrolyzed rapidly in alkaline solution (half lives (t_1/2)<one day), were stable in solutions of acetonitrile, tetrahydrofuran and diluted hydrochloric acid (t_1/2>five months) and were unstable in solutions of methanol and ethanol (t_1/2<one month). These alkaloids were separated on an octadecylsilica column with isocratic elution using a solvent mixture of tetrahydrofuran and 0.2% trifluoroacetic acid (14:86, v/v), which was found to be the optimal solvent of the elution systems examined. Calibration curves with UV detection were linear on injection of amounts ranging from 2.5 to 500 ng, and the limit of detection was 1 ng (S/N = 3). These four alkaloids in aqueous solution were recovered almost totally by solid-phase extraction using the styrene polymer resin, Sep-Pak Plus PS-1, and were eluted using a mixture of acetonitrile and hydrochloric acid. These Aconitum alkaloids were confirmed by HPLC coupled with fast atom bombardment MS, giving their protonated molecular ions as base peaks. These alkaloids were detected by HPLC with UV detection from blood samples spiked with more than 50 ng ml⁻¹ of alkaloids, but were not detectable from urine samples spiked with 5 μg ml⁻¹ of alkaloids because of severe sample interference.     

Selective high-performance liquid chromatographic determination of artesunate and α- and β-dihydroartemisinin in patients with falciparum malaria

A novel solid-phase extraction and a robust high-performance liquid chromatographic (HPLC) separation procedure for artesunate and α- and β-dihydroartemisinin, using post-column alkali decomposition and UV detection, is described. Extraction was performed with Bond-Elut Phenyl solid-phase extraction cartridges and analysis by HPLC was carried out using a Waters Symmetry C₈ 5-μm 150 × 3.9 mm I.D. column. The mobile phase was 50% acetonitrile in 0.1 M acetate buffer (pH 4.8) delivered at a flow-rate of 0.7 ml/min. The column eluate was mixed with 1.2 M potassium hydroxide in 90% methanol delivered at 0.3 ml/min, in a 1-ml reaction coil at 69°C, to form UV-absorbing chromophores which were detected at 290 mm. The recovery of all analytes was greater than 80%. There was no significant difference in the peak-area ratio of α- and β-dihydroartemisinin in plasma. Preliminary pharmacokinetic data from six adult Vietnamese patients who received 120 mg of artesunate by intravenous injection for the treatment of acute falciparum malaria are presented. Despite limited data, the mean half-life of artesunate was approximately 3.5 min while that for dihydroartemisinin was 34 min. These data confirm the relatively rapid clearance of both artesunate and its principal active metabolite, dihydroartemisinin.     

Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica

A rapid procedure for the efficient extraction of prostaglandins, thromboxanes and hydroxy fatty acids from urine, plasma and tissue homogenates has been developed. Fractions containing these substances are acidified and passed through a column of octadecylsilyl silica, which retains oxygenated metabolites of arachidonic acid. Phospholipids, proteins and very polar materials either are not retained or can be eluted with dilute aqueous ethanol. Nonpolar lipids and monohydroxy fatty acids are then eluted with petroleum ether or benzene. Subsequent elution of the column with methyl formate gives a fraction containing prostaglandins and thromboxanes which is much less contaminated with extraneous material than that obtained by conventional extraction of aqueous media with organic solvents. The methyl formate can be removed rapidly under a stream of nitrogen and the components of the sample purified directly by high pressure liquid chromatography (HPLC). An improved method for the purification of prostaglandins and TXB₂ by HPLC on silica columns is reported.     

Determination of diphenylpyraline in plasma and urine by high-performance liquid chromatography

A rapid, reliable and specific reversed-phase high-performance liquid chromatographic procedure is described for the determination of diphenylpyraline hydrochloride at nanogram concentrations in plasma and urine. After extraction of the drug with n-pentane-2-propanol (50:1) from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with methanol and chromatographed using a 5-μm Asahipak ODP-50 C₁₈ column with UV detection at 254 nm. The elution time for diphenylpyraline was 7.9 min. The overall recovery of diphenylpyraline from spiked plasma and urine samples at concentrations ranging from 53 to 740 ng/ml were 94.65% and 92.29%, respectively. Linearity and precision data for plasma and urine standards after extraction were acceptable. The limit of detection was 15 ng/ml for both plasma and urine samples at 0.002 AUFS.     

HPLC method for determination of verapamil in human plasma after solid-phase extraction

A simple, rapid and precise HPLC method has been developed for the assay of verapamil in human plasma. The clean up of the plasma samples was tested using several adsorbents for solid-phase extraction and best recovery was obtained using mixed-mode cartridges (HLB - hydrophilic-lipophilic balance) ranging between 94.70 and 103.71%. HPLC separation was performed with isocratic elution on Lichrospher 60 RP-select B column (250 mm × 4 mm I.D., 5 μm particle size). The mobile phase was 40% acetonitrile and 0.025 mol/L KH₂PO₄ with pH 2.5 at flow rate of 1 mL/min. Diltiazem was used as internal standard and the detection wavelength was 200 nm. The calibration curves were linear in the range of 10–500 ng/mL. The developed method is convenient for routine analysis of verapamil in human plasma.     

Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography

A simple, rapid and reproducible reversed-phase high-performance liquid chromatographic method for the simultaneous determination of benzoic acid (BA), phenylacetic acid (PAA) and their respective glycine conjugates hippuric acid (HA) and phenaceturic acid (PA) in sheep urine is described. The procedure involves only direct injection of a diluted urine sample, thus obviating the need for an extraction step or an internal standard. The compounds were separated on a Nova-Pak C₁₈ column with isocratic elution with acetate buffer (25 mM, pH 4.5)—methanol (95:5). A flow-rate of 1.0 ml/min, a column temperature of 35°C and detection at 230 nm were employed. These conditions were optimized by investigating the effects of pH, molarity, methanol concentration in the mobile phase and column temperature on the resolution of the metabolites. The total analysis time was less than 15 min per sample. At a signal-to-noise ratio of 3 the detection limits for ten-fold diluted urine were 1.0 μg/ml for BA and HA and 5.0 μg/ml for PAA and PA with a 20-μl injection.     

Determination of phenprocoumon, warfarin and their monohydroxylated metabolites in human plasma and urine by liquid chromatography-mass spectrometry after solid-phase extraction

A high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for the quantification of phenprocoumon, warfarin, and their known monohydroxylated metabolites in human plasma and urine was developed using a simple, selective solid-phase extraction scheme. Chromatographic separation was achieved on a reversed-phase Luna C18 column and step gradient elution resulted in a total run time of about 13 min. Limits of quantification (LOQ) were < or = 40 nM for the parent compounds and < or = 25 nM for the metabolites and the limit of detection (LOD) was < or = 2.5 nM for all analytes. Average recovery was 84% (+/- 3.7) and 74% (+/- 13.2) in plasma and urine, respectively. Intra- and inter-day coefficients of variation were < or = 8.6 and < or = 10.6% in plasma and urine, respectively. The method was successfully applied to the analysis of phenprocoumon samples from four healthy volunteers and should prove useful for future comparative studies of warfarin and phenprocoumon pharmacokinetics.     

SPE-GC-MS for the sampling and determination of unmetabolized styrene in urine

The urinary excretion of unmetabolized styrene can be a very good indicator for biomonitoring styrene in occupationally exposed people. The use of a new urine sampling system, involving a solid-phase extraction cartridge, offers several advantages for determining styrene. The advantages are especially related to the pre-analytical phase of styrene determination, which may be influenced by many variables. The effect on styrene recovery of sorbent type, eluting solvent, elution volume, elution flow-rate, and the addition of methanol to the washing solvent, was evaluated by experimental design methodology. As a result, Oasis HLB cartridges were selected for urine sampling, as well as 1.5 mL of ethyl acetate at 0.5 mL/min for eluting the retained styrene. These conditions were then applied to the validation of the solid-phase extraction combined with GC-MS method for the sampling and analysis of unmetabolized styrene in urine. The overall uncertainty was in the 12-22% range and the limit of detection was 2.2 microg/L for a 4 mL urine sample. The stability of styrene has been studied both in cartridges and in vials under different storage periods. After 1 month period the styrene stored on cartridges at room temperature remained stable, whereas this is not the case for styrene recovery from vials. The results obtained indicate that on-site solid-phase extraction of urine can provide a simple, accurate and reproducible sampling and analytical method for the biomonitoring of styrene in urine.     

Simple and sensitive procedure for the assay of serotonin and catecholamines in brain by high-performance liquid chromatography using fluorescence detection

A simple, rapid and specific method for the determination of serotonin and catecholamines in brain is described. After tissue homogenisation, catecholamines are isolated by adsorption onto alumina and elution with perchloric acid. Serotonin is isolated by extraction into n-heptanol and back-extraction into acid. High-performance liquid chromatography of the acid extracts is performed with a C₁₈ reversed-phase column and simple mobile phases. Detection is by the intrinsic fluorescence of the amines on excitation at 200 nm. Detection limits are 100 pg for norepinephrine, 300 pg for dopamine and 20 pg for serotonin. The results are found to correlate well with a catechol O-methyl transferase radioenzymatic assay for catecholamines and a ninhydrin derivatisation procedure for serotonin.