Rapid and direct analysis of gamma-hydroxybutyric acid in urine by capillary electrophoresis-electrospray ionization ion-trap mass spectrometry

The present work was aimed at the development of a capillary electrophoretic analysis of gamma-hydroxybutyric acid (GHB) using electrospray ion trap mass spectrometry to achieve the direct and unequivocal detection of this analyte in human urine. Optimized capillary electrophoretic conditions were: injection, 20 s at 0.5 psi (1 psi = 6894.76 Pa); buffer electrolyte, 12.5 mM ammonium formate adjusted to pH 8.35 with diethylamine; fused silicacapillary: 100 cm x 50 microm i.d.; separation voltage, 25 kV (forward polarity) + 0.5 psi; room temperature. Electrospray and mass spectrometric conditions were: drying gas and nebulizing gas (nitrogen) at flow rate 3 l/min, temperature 250 degrees C, nebulizer pressure: 10 psi; sheath liquid solution: methanol-water (90:10) containing 0.1% ammonia delivered at 3 microl/min; spray voltage 3.5 kV. Mass spetrometric detection was carried out in the selected ion monitoring mode of negative molecular ions at 103 m/z for GHB and 115 m/z for maleic acid (I.S.). Under these conditions the baseline separation of GHB and the I.S. was obtained. The selectivity of the analysis allowed for direct injection of unextracted urine, previously diluted 1:4 with water. Linearity was assessed in the GHB concentration range from 80 to 1280 microg/ml in urine. Analytical sensitivity (as limit of detection) resulted about 5 microg/ml in water and 20 microg/ml in original urine. Analytical precision was fairly acceptable with R.S.D. values lower than 5% for migration times and 18% for quantitation in real samples, in both intra day and day-to-day experiments. On these grounds, the developed method can be adopted for rapid identification of acute intoxications from GHB in humans.     

Determination of gamma-hydroxybutyric acid (GHB) in plasma and urine by headspace solid-phase microextraction and gas chromatography/positive ion chemical ionization mass spectrometry

A new method for the qualitative and quantitative analysis of gamma-hydroxybutyric acid (GHB) in plasma and urine samples is described. It involves the conversion of GHB to gamma-butyrolactone (GBL), its subsequent headspace solid-phase microextraction (SPME), and detection by gas chromatography/positive ion chemical ionization mass spectrometry (GC/PICI-MS), using D(6)-GBL as internal standard. The assay is linear over a plasma GHB range of 1-100 microg/mL (n = 5, r = 0.999) and a urine GHB range of 5-150 microg/mL (n = 5, r = 0. 998). Relative intra- and inter-assay standard deviations, determined for plasma and urine samples at 5 and 50 microg/mL, are all below 5%. The method is simple, specific and reasonably fast. It may be applied for clinical and forensic toxicology as well as for purposes of therapeutic drug monitoring.     

Carbon isotopic ratio analysis by gas chromatography/combustion/isotope ratio mass spectrometry for the detection of gamma-hydroxybutyric acid (GHB) administration to humans

Since GHB (gamma-hydroxybutyric acid) is naturally produced in the human body, clinical and forensic toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. To suggest an alternative to the use of interpretative concentration cut-offs, the detection of exogenous GHB in urine specimens was investigated by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GHB was isolated from urinary matrix by successive purification on Oasis MCX and Bond Elute SAX solid-phase extraction (SPE) cartridges prior to high-performance liquid chromatography (HPLC) fractioning using an Atlantis dC18 column eluted with a mixture of formic acid and methanol. Subsequent intramolecular esterification of GHB leading to the formation of gamma-butyrolactone (GBL) was carried out to avoid introduction of additional carbon atoms for carbon isotopic ratio analysis. A precision of 0.3 per thousand was determined using this IRMS method for samples at GHB concentrations of 10 mg/L. The (13)C/(12)C ratios of GHB in samples of subjects exposed to the drug ranged from -32.1 to -42.1 per thousand, whereas the results obtained for samples containing GHB of endogenous origin at concentration levels less than 10 mg/L were in the range -23.5 to -27.0 per thousand. Therefore, these preliminary results show that a possible discrimination between endogenous and exogenous GHB can be made using carbon isotopic ratio analyses.     

A new method for the HPLC determination of gamma-hydroxybutyric acid (GHB) following derivatization with a coumarin analogue and fluorescence detection: application in the analysis of biological fluids

A new method of the determination of gamma-hydroxybutyric acid (GHB) in human biological fluids cerebrospinal fluid (CSF) and saliva after off-line derivatization is described. The proposed method was based on the reaction of 4-bromomethyl-7-methoxy coumarin (Br-MMC) with GHB in the presence of dibenzo-18-crown-6-ether (acting as reaction catalyst) to produce a fluorescent derivative. The formed derivative was monitored fluorimetrically at lambda(ext.)=330 nm and lambda(em.)=390 nm. The effect of derivatization parameters such as the concentration of Br-MMC, reaction time and the temperature was investigated in order to achieve the maximum method's sensitivity. The separation was achieved by use of a C(18) analytical column (Kromasil 250 mm x 4 mm i.d., 5 microm) while the injected sample volume was set to 25 microL. A binary gradient elution program of methanol versus phosphate buffer (40 mM, pH 3) was selected for the quantitative analysis of GHB. The method showed satisfactory linearity (R(2)=0.9979) in a linear range from 2.4 x 10(-6) to 7.2 x 10(-5) M. Ultrafiltration method was employed for the pre-treatment of the cerebrospinal fluid (CSF) prior to the analysis of GHB. The limit of detection (LOD) of the method was 3 x 10(-7) M in saliva and 2 x 10(-7) M in CSF samples, respectively, while the limit of quantitation (LOQ) was 1 x 10(-6) M for both specimens. The proposed protocol offers sensitivity comparing with the existing HPLC analytical methods or the CE indirect UV methods and can function as an attractive alternative to be used in clinical and toxicological analysis.     

Capillary electrophoresis of urinary porphyrins with absorbance and fluorescence detection

Urinary porphyrins are separated in a 72 cm x 50 microns I.D. fused-silica capillary by micellar electrokinetic capillary chromatography with 100 mM sodium dodecyl sulfate and 20 mM 3-(cyclohexylamino)-1-propanesulfonic acid at pH 11. Detection is accomplished by absorbance at 400 nm or fluorescence with excitation at 400 nm and emission at wavelengths above 550 nm. Substantial trace enrichment is found for porphyrins in urine samples or for porphyrin standards prepared without surfactant in the injection buffer. Limits of detection are in the 100 pmol/ml concentration range with an optimized fluorescence system. The method is shown suitable for the determination of porphyrins in clinical urine specimens. Comparisons are made between electrophoretic and chromatographic methods for the separation and detection of urinary porphyrins.     

Simultaneous analysis of gamma-hydroxybutyric acid and its precursors in urine using liquid chromatography-tandem mass spectrometry

We have developed a rapid method that enables the simultaneous analysis of gamma-hydroxybutyrate (GHB) and its precursors, i.e. gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in urine. The method comprised a simple dilution of the urine sample, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatographic separation was achieved using an Atlantis dC18 column, eluted with a mixture of formic acid and methanol. The method was linear from 1-80 mg/L for GHB and 1,4-BD and from 1-50 mg/L for GBL. The limit of quantification was 1 mg/L for all analytes. The procedure, which has a total analysis time (including sample preparation) of less than 12 min, was fully validated and applied to the analysis of 182 authentic urine samples; the results were correlated with a previously published GC-MS procedure and revealed a low prevalence of GHB-positive samples. Since no commercial immunoassay is available for the routine screening of GHB, this simple and rapid method should prove useful to meet the current increased demand for the measurement of GHB and its precursors.     

Evaluation of microchip capillary electrophoresis with external contactless conductivity detection for the determination of major inorganic ions and lithium in serum and urine samples

The determination of inorganic ions in clinical samples in less than 90 seconds was demonstrated for microchip capillary electrophoresis using capacitively coupled contactless conductivity detection (C(4)D). Bare electrophoresis chips were used in combination with external electrodes which were part of the chip holder. In order to achieve the required selectivity and sensitivity, an optimization of the electrode layout was carried out. Limits of detection (LOD) of 1 microM for K(+), 1.5 microM for Ca(2+), 3 microM for Na(+), 1.75 microM for Mg(2+) and 7.5 microM for Li(+) were achieved. The determination of inorganic cations (NH(4)(+), K(+), Na(+), Ca(2+), Mg(2+)) and anions (Cl(-), NO(3)(-), SO(4)(2-), phosphate) in blood serum and urine samples was possible in one common electrolyte solution containing 15 mM L-arginine, 10.75 mM maleic acid and 1.5 mM 18-crown-6 at pH 5.90 by simply switching the separation voltage from positive to negative polarity. Lithium, present at significant levels when used for therapeutic purposes, can also be determined in blood serum using a slightly modified background electrolyte solution.     

Determination of γ‐hydroxybutyric acid in saliva by capillary electrophoresis coupled with contactless conductivity and indirect UV absorbance detectors

The aim of the current study was to optimise and validate the methodology for determination of γ‐hydroxybutyric acid (GHB) in saliva by CE combined with a contactless conductivity detector (C⁴D) and indirect UV absorbance detection (λ_ABS = 210 nm). The optimized BGE, consisting of 8.5 mM maleic acid, 17 mM arginine, 255 μM cetyltrimethylammonium bromide (CTAB), and 15% acetonitrile, was evaluated for the separation of GHB in saliva within 6 min. The performance characteristics of the CE‐C⁴D‐indirect UV methodology was validated. The instrument detection and quantification limits were 0.49 and 1.6 mg/L for C⁴D, and 5.1 mg/L and 17.0 mg/L for indirect UV, respectively. The linearity was obtained over the range from 2.5 to 400 mg/L for C⁴D and from 12.5 to 400 mg/L for indirect UV. The interday precisions were within 2.3–5.7% and intraday precisions were within 1.6–9.0% for C⁴D as well as 2.1–9.3%, 5.6–10.1% for indirect UV in spiked saliva, respectively. The recoveries were within 87.2–104.4%. The matrix effects were +53.2% for small concentrations up to 25 mg/L for C⁴D and +23.6% for concentrations up to 75 for mg/L for indirect UV detection. No matrix effects were observed for higher concentration levels. In conclusion, CE‐C4D‐indirect UV can offer a rapid, accurate, sensitive, and definitive method for the determination of GHB abuse in saliva samples as a forensic screening tool.     

Screening and confirmation methods for GHB determination in biological fluids

The purpose of this review is to provide a comprehensive overview of reported methods for screening and confirmation of the low-molecular-weight compound and drug of abuse gamma-hydroxybutyric acid (GHB) in biological fluids. The polarity of the compound, its endogenous presence, its rapid metabolism after ingestion, and its instability during storage (de novo formation and interconversion between GHB and its lactone form gamma-butyrolactone) are challenges for the analyst and for interpretation of a positive result. First, possible screening procedures for GHB are discussed, including colorimetric, enzymatic, and chromatography-based procedures. Confirmation methods for clinical and forensic cases mostly involve gas chromatography (coupled to mass spectrometry), although liquid chromatography and capillary zone electrophoresis have also been used. Before injection, sample-preparation techniques include (a combination of) liquid–liquid, solid-phase, or headspace extraction, and chemical modification of the polar compound. Also simple “dilute-and-shoot” may be sufficient for urine or serum. Advantages, limitations, and trends are discussed.     

Determination of the pyridinium metabolite derived from haloperidol in brain tissue, plasma and urine by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective method for the determination of the pyridinium metabolite (HPP+) derived from the antipsychotic drug haloperidol (HP) in brain tissue, plasma and urine using high-performance liquid chromatography with fluorescence detection is described. The HPP+ present in biological samples was extracted using a Sep-Pak C18 cartridge. Recoveries of HPP+ ranged from 78 to 90%. Final separation and quantitative estimations of HPP+ were achieved on a C18 reversed-phase column employing a mobile phase of acetonitrile-30 mM ammonium acetate (40:60, v/v) containing 10 mM triethylamine and adjusted to pH 3 with trifluoroacetic acid. The fluorescence detection utilized an excitation wavelength of 304 nm and an emission wavelength of 374 nm. Standard curves were linear in the range of 2.5-100 ng/ml for brain tissue homogenate and plasma samples and 10-500 ng/ml for urine samples. The detection limit of HPP+ was about 1 ng/ml in all biological samples. The concentrations of HPP+ in brain tissue, plasma and urine from HP-treated rats were determined using this method.     

Determination of ethyl sulfate--a marker for recent ethanol consumption--in human urine by CE with indirect UV detection

A CE method for the determination of the ethanol consumption marker ethyl sulfate (EtS) in human urine was developed. Analysis was performed in negative polarity mode with a background electrolyte composed of 15 mM maleic acid, 1 mM phthalic acid, and 0.05 mM cetyltrimethylammonium bromide (CTAB) at pH 2.5 and indirect UV detection at 220 nm (300 nm reference wavelength). This buffer system provided selective separation conditions for EtS and vinylsulfonic acid, employed as internal standard, from urine matrix components. Sample pretreatment of urine was minimized to a 1:5 dilution with water. The optimized CE method was validated in the range of 5-700 mg/L using seven lots of urine. Intra- and inter-day precision and accuracy values, determined at 5, 60, and 700 mg/L with each lot of urine, fulfilled the requirements according to common guidelines for bioanalytical method validation. The application to forensic urine samples collected at autopsies as well as a successful cross-validation with a LC-MS/MS-based method confirmed the overall validity and real-world suitability of the developed expeditious CE assay (sample throughput 130 per day).     

Determination of ammonia, creatinine and inorganic cations in urine using CE with contactless conductivity detection

CE with capacitively coupled contactless conductivity detection (C(4)D) was used to determine waste products of the nitrogen metabolism (ammonia and creatinine) and of biogenic inorganic cations in samples of human urine. The CE separation was performed in two BGEs, consisting of 2 M acetic acid + 1.5 mM crown ether 18-crown-6 (BGE I) and 2 M acetic acid + 2% w/v PEG (BGE II). Only BGE II permitted complete separation of all the analytes in a model sample and in real urine samples. The LOD values for the optimized procedure ranged from 0.8 microM for Ca(2+) and Mg(2+) to 2.9 microM for NH(4)(+) (in terms of mass concentration units, from 7 microg/L for Li(+) to 102 microg/L for creatinine). These values are adequate for determination of NH(4)(+), creatinine, Na(+), K(+), Ca(2+) and Mg(2+) in real urine samples.     

Pulsed electrochemical detection of orotic acid by an activated potential waveform at a gold working electrode following anion-exchange chromatography

The application of activated pulsed amperometric detection (APAD) for the determination of orotic acid (OrA) in real samples at a gold working electrode in alkaline solutions, in combination with anion-exchange chromatography, is reported. Such an activated potential waveform was designed with an initial step that involves the formation of redox active species (e.g., adsorbed AuOH/AuO), which in turn is halted upon lowering the applied potential at the detection value while the adsorbed gold hydroxide/oxide species are still catalytically active. A direct comparison between the activated potential waveform and the more commonly used pulsed amperometric detection showed roughly a 20-fold increase in sensitivity. The chromatographic separation of OrA was accomplished by using a microbore anion-exchange column eluted with an isocratic mobile phase composed of 100 mM NaOH+40 mM NaNO(3). Orotic acid was determined at the concentration ranges of 0.2-30 microM (r=0.9997) with an absolute detection limit of 80 pg (10 microL injected). The levels of OrA in cows' milk samples evaluated by standard additions, using 5-aminoorotic acid as an internal standard, ranged from 56 to 126 mg/L. Lower levels were found in raw sheep's milk (<20 mg/L). The assay is shown to be very useful in clinical investigations where relatively high levels of OrA in human urine are correlated to metabolic diseases.     

Determination of oxalic acid in urine by co-electroosmotic capillary electrophoresis with amperometric detection

Co-electroosmotic capillary electrophoresis with amperometric detection at a Cobalt phthalocyanine (CoPC) modified carbon paste electrode was evaluated for the determination of oxalic acid in urine. The running buffer consisted of 10 mM phosphate (pH=5.70) and 0.25 mM Cetyltrimethylammonium bromide. Under the optimum conditions, a detection limit of 0.12 muM was achieved for oxalic acid. The response was linear between 0.5 and 1000 muM with a correlation coefficient of 0.9995. Applications of the method to real urine samples were described.     

Determination of vigabatrin in human plasma and urine by high-performance liquid chromatography with UV-Vis detection

A simple and reliable high-performance liquid chromatographic (HPLC) method with UV-Vis detection has been developed and validated for the determination of vigabatrin (VG) in human plasma and urine. The samples were pre-column derivatizated with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS). A good chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 10 mM orthophosphoric acid (pH 2.5) gradient elution. Tranexamic acid was used as an internal standard (I.S.). The method was linear over the concentration range of 0.8-30.0 microg/ml for both samples. The method is precise (relative standard deviation (R.S.D.) <9.13%) and accurate (relative mean error (RME) <-8.75%); analytical recoveries were 81.07% for plasma and 83.05% for urine. The assay was applied to pharmacokinetic study in a healthy volunteer after a single oral administration of 1 g of vigabatrin.     

Capillary zone electrophoresis applied to the determination of the angiotensin-converting enzyme inhibitor cilazapril and its active metabolite in pharmaceuticals and urine

A capillary zone electrophoresis method has been developed for the quantitation of antihypertensive drug cilazapril and its active metabolite cilazaprilat in pharmaceuticals and urine. The separation of the compounds was performed in a fused-silica capillary filled with the running electrolyte, which consisted of a 60 mM borate buffer solution at pH 9.5. Under the optimized experimental conditions, the separation took less than 5 min. The analysis of urine samples required a previous solid-phase extraction step using C8 cartridges. The method was successfully applied to the determination of the drug and its metabolite in urine samples obtained from three hypertensive patients (detection limits of 115 ng ml(-1) for cilazaprilat and 125 ng ml(-1) for cilazapril) and to pharmaceutical dosage forms. The method was validated in terms of reproducibility, linearity and accuracy.     

Determination of chlorophenols in human urine based on the integration of on-line automated clean-up and preconcentration unit with micellar electrokinetic chromatography.

A new method was developed and validated for the determination of chlorophenols in human urine by using micellar electrokinetic chromatography (MEKC) coupled via a mechanic arm to an on-line automatic clean-up and preconcentration unit for urine samples. Separation is accomplished by using a selective buffer consisting of 15 mM borate, 25 mM phosphate and 100 mM sodium dodecyl sulfate (SDS) at pH 9.1 in addition to a positive power supply of 25 kV at 18 degrees C. The proposed capillary electrophoresis (CE) method allows the separation of 11 chlorophenols within 7 min with a reproducibility as relative standard deviation (RSD) between 2.6% and 7.2%, and limits of detection (LODs) between 0.08 and 0.46 microg/mL for all chlorophenols. Urine samples were previously hydrolyzed with 37% HCl at 80 degrees C for 60 min and then cleaned on a C-18 mini-column. Recoveries ranged from 58% to 103%. The preconcentration treatment affords limits of determination between 4 and 12 ng/mL for all chlorophenols except pentachlorophenol and 4-chlorophenol, which could not be determined. The overall analysis time, including on-line clean-up, preconcentration and electrophoretic separation is 20 min per sample.     

Rapid and simple pretreatment of human body fluids using electromembrane extraction across supported liquid membrane for capillary electrophoretic determination of lithium

Electromembrane extraction was used for simultaneous sample cleanup and preconcentration of lithium from untreated human body fluids. The sample of a body fluid was diluted 100 times with 0.5 mM Tris solution and lithium was extracted by electromigration through a supported liquid membrane composed of 1-octanol into 100 mM acetic acid acceptor solution. Matrix compounds, such as proteins, red blood cells, and other high-molecular-weight compounds were efficiently retained on the supported liquid membrane. The liquid membrane was anchored in pores of a short segment of a polypropylene hollow fiber, which represented a low cost, single use, disposable extraction unit and was discarded after each use. Acceptor solutions were analyzed using capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C(4) D) and baseline separation of lithium was achieved in a background electrolyte solution consisting of 18 mM L-histidine and 40 mM acetic acid at pH 4.6. Repeatability of the electromembrane extraction-CE-C(4) D method was evaluated for the determination of lithium in standard solutions and real samples and was better than 0.6 and 8.2% for migration times and peak areas, respectively. The concentration limit of detection of 9 nM was achieved. The developed method was applied to the determination of lithium in urine, blood serum, blood plasma, and whole blood at both endogenous and therapeutic concentration levels.     

Determination of folic acid by capillary electrophoresis with chemiluminescence detection

A rapid and simple method is presented for the determination of folic acid (FA) by capillary electrophoresis (CE) with chemiluminescence (CL) detection. This method was based on enhance effect of FA on the CL reaction between luminol and BrO(-) in alkaline aqueous solution. Optimal separation and determination was obtained with an electrophoretic buffer of 35 mM sodium borate (pH 9.4) containing 0.8 mM luminol, and an oxidizer solution of 1.6 mM NaBrO in 100 mM NaCO(3) buffer solution (pH 12.0). Under the optimal conditions, the determination of FA was achieved in less than 20 min, and the detection limit was 2.0 x 10(-8) M (S/N=3). The relative standard deviations (RSDs) on peak area and migration time were in the 1.5 and 1.1%, respectively. The present CE-CL method was applied to the determination of FA in commercial pharmaceutical tablets, apple juices and human urine.     

Separation of fifteen quinolones by high performance liquid chromatography: application to pharmaceuticals and ofloxacin determination in urine

A simple chromatographic method is described for assaying 15 quinolones and fluoroquinolones (pipemidic acid, marbofloxacin, enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, lomefloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, nalidixic acid, flumequine and piromidic acid), in urine and pharmaceutical samples. The determination was achieved by LC using an RP C18 analytical column. A mobile phase composed of mixtures of methanol-ACN-10 mM citrate buffer at pH 3.5 and 10 mM citrate buffer at pH 4.5, delivered under an optimum gradient program, at a flow rate of 1.5 mL/min, allows to accomplish the chromatographic separation in 26 min. For detection, diode-array UV-Vis at 280 nm and fluorescence detection set at excitation wavelength/emission wavelength: 280/450, 280/ 495, 280/405 and 320/360 nm were used. Detection and quantification limits were between 0.3-18 and 0.8-61 ng/mL, respectively. The method was validated in terms of interday (n = 6) and intraday (n = 6) precision and accuracy. The procedure was successfully applied to the analysis of human and veterinary pharmaceuticals. Also, ofloxacin was determined in human urine samples belonging to a patient undergoing treatment with this active principle, among others.