Rapid assay of cocaine, opiates and metabolites by gas chromatography—mass spectrometry

The simultaneous assay of cocaine, opiates and metabolites in small biological samples continues to be a difficult task. This report focuses upon tabulation of important techniques (extraction, derivatization, chromatographic conditions, detection mode, data acquisition) reported over the last decade that were used in the development of assays for these analytes. The most prevalent procedures for extraction of cocaine, opiates and metabolites were liquid—liquid and solid-phase extraction isolation methods. Following extraction analytes were derivatized and analyzed by gas chromatography—mass spectrometry. The technique most often used for chromatographic separation was fused-silica capillary column gas chromatography. Detection generally was performed by selected ion monitoring in the positive-ion electron-impact ionization mode, although full-scan acquisition and positive- and negative-ion chemical ionization methods have been used. It was apparent from the review that there is a continuing need for greater sensitivity and selectivity in the assay of highly potent opiates and for cocaine and metabolites.     

Quantitation of 2-chlorovinylarsonous acid in human urine by automated solid-phase microextraction--gas chromatography--mass spectrometry

Lewisite [dichloro(2-chlorovinyl)arsine] is a highly toxic chemical warfare agent with vesicant properties. The accidental exposure to lewisite or its intentional use as a chemical terrorism weapon are a public health threat and warrant investigations for the development of analytical methods to detect biomarkers of exposure to lewisite. Under aqueous conditions, lewisite rapidly hydrolyzes to the non-volatile 2-chlorovinylarsonous acid (CVAA). We have developed a sensitive, simple, and automated method for measuring CVAA in human urine. The assay is based on the use of solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) after derivatization of the CVAA with 1,3-propanedithiol (PDT). The volatile CVAA-PDT is adsorbed onto a SPME fiber and analyzed by GC-MS. The assay was validated on human urine samples spiked with CVAA to determine the accuracy, precision, and limit of detection (LOD). The LOD was 7.4 pg in 1 ml of urine.     

Determination of free and total (free plus protein-bound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with fluorescence detection

A simple, sensitive and accurate method for the estimation of free and total (free plus protein-bound) melatonin (MLT) in human plasma and cerebrospinal fluid (CSF) is described. Via Chem-Elut cartridges, free and total MLT (the latter obtained after a deproteinization step) were quantified in dichloromethane-extracted samples and analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with fluorimetric detection. The column used was an Extrasil ODS-2 (3 microm, 150 x 4.6 mm I.D.), while the mobile phase consisted of 75 mM sodium acetate-acetonitrile (72:28, v/v) (pH 5.0). Repeatability and reproducibility of the method were 3.24 and 9.4%, respectively. The recovery of melatonin from plasma and CSF was 99.9+/-4.0% for non-deproteinized samples and 93.2+/-4.8% for deproteinized samples. The detection limit of the assay was 0.5 pg/ml. In human plasma, the mean+/-SD concentrations in the darkness period were 23.18+/-7.44 pg/ml for free melatonin and 82.5+/-36.48 pg/ml for total melatonin, while the lowest concentrations detected during daytime were 2.23+/-2.22 and 7.40+/-5.68 pg/ml, respectively. Detection of MLT in CSF was 5.01+/-2.31 and 28.55+/-6.95 pg/ml for the free and total fraction, respectively.     

Rapid detection and identification of biological and chemical agents by immunoassay, gene probe assay and enzyme inhibition using a silicon-based biosensor

A rapid biosensor assay procedure that utilizes biotin streptavidin mediated filtration capture onto nitrocellulose membrane, in conjunction with a silicon-based light-addressable potentiometric sensor (LAPS) was developed for detection and identification of biological and chemical threat agents. Sandwich immunoassays, nucleic acid hybridization assays and enzyme inhibition assays are described. For immunoassays, the lower limits of detection (LOD) per 100-microl sample were approximately 5 pg/ml for protein (Staphylococcal enterotoxin B), 2 ng/ml for virus (Newcastle disease virus), and 20 ng/ml for vegetative bacteria (Brucella melitensis). In a dual gene probe assay format, the LOD was 0.30 fmol (1.8 x 10(8) copies per 60-microl) of single stranded target DNA. Enzyme inhibition assays on the LAPS using acetylcholinesterase were able to detect soman and sarin in aqueous samples at 2 and 8 pg (100 and 600 pM), respectively. The assays were easy to perform and required a total time equal to the reaction period plus about 15 min for filtering, washing and sensing. The assay format is suitable for detection of a wide range of infectious and toxic substances. New assays can be developed and optimized readily, often within 1 or 2 days.     

High-performance liquid chromatographic method with electrochemical detection for the analysis of O6-methylguanine

An improved system consisting of a combination of high-performance liquid chromatographic methods with electrochemical detection for the separation and analysis of the DNA adduct O⁶-methylguanine (O6MG) has been developed. This adduct is produced by the interaction of methylating agents with DNA and induces mispairing in the DNA of the target cells. A good separation of modified from unmodified bases is first achieved with an HPLC system using a Partisil 10 SCX column and a salt gradient. A second HPLC step with electrochemical detection and a C18 column is used for farther separation and quantitation of O⁶-methylguanine. This method shows a linear response up to 15 pg of O6MG tested. The lowest amount detected was 0.5 pg of O6MG and is highly reproducible. This method is useful to study DNA damage as a product of cellular metabolism and its effects on the process of carcinogenesis.     

Column-switching high-performance liquid chromatographic analysis of BO-2727, a new carbapenem antibiotic, in human plasma and urine by direct injection

A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05–50 μg/ml in plasma and 0.5–100 μg/ml in urine. The limits of quantification for plasma and urine were found to be 0.05 μg/ml and 0.5 μg/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.     

Drug analysis by direct liquid introduction micro liquid chromatography mass spectrometry

The analytical capabilities of a micro high performance liquid chromatograph interfaced to an unchanged quadrupole mass spectrometer are presented. Continuous monitoring of the total micro liquid chromatographic effluent allows full scan chemical ionization mass spectra of from one to five nanograms of drugs and their metabolites to be recorded. The interface is a simple, inexpensive device which can be assembled from commercially available components. An eight microliter per minute flow rate of the micro liquid chromatographic eluant allows separation and identification of biologically important substances not amenable to gas chromatography mass spectrometry techniques. The sensitivity of micro liquid chromatography mass spectrometry performed as described is comparable with gas chromatography mass spectrometry and is achieved by introducing the total micro liquid chromatographic effluent into the chemical ionization ion source of the mass spectrometer. Selected ion monitoring provides 20 pg detection limits of phenothiazine tranquilizers injected on column.     

Quantitation of cocaine and cocaethylene in canine serum by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic procedure for the determination of cocaine and cocaethylene in canine serum has been developed. The compounds were extracted from 1 ml of alkalinized canine serum with hexane. Chromatographic separation was achieved with a cyanopropyl column (250 × 4.6 mm I.D., 5 μm) using a mobile phase of acetonitrile and phosphate buffer, pH 7.40 (38:62, v/v) flowing at 1 ml/min. Eluate was monitored by a variable-wavelength UV detector set to 230 nm. The extraction procedure yields an average recovery of 99 and 96% for cocaine and cocaethylene, respectively. The between-day coefficients of variation, at 2400 ng/ml, for cocaine and cocaethylene were both 8.6% and the within-day coefficients of variation, at 400 ng/ml, for cocaine and cocaethylene were 7.3 and 8.0%, respectively. A concentration-time profile resulting from administration of 3 mg/kg cocaine and cocaethylene to the dog revealed a similar disposition between cocaine and cocaethylene, with a clearance and volume of distribution at steady-state values of 72.8 and 61.0 ml/min/kg and 2.6 and 2.7 1/kg, respectively.     

Determination of cocaine and its metabolites in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating cocaine and its three metabolites in rat serum microsamples (50 μl). The separation used a 2.1-mm I.D. reversed-phase Brownlee C₁₈ column with an isocratic mobile phase consisting of methanol–acetonitrile–25.8 mM sodium acetate buffer, pH 2.2, containing 1.29·10⁻⁴M tetrabutylammonium phosphate (12.5:10:77.5, v/v/v). The detection limit was 2.5 ng/ml for all the compounds using an ultraviolet detector operated at 235 nm. The method was used to study the pharmacokinetics of cocaine after an intravenous (i.v.) bolus dose (4 mg/kg).     

Determination of diphenylmethane antihistaminic drugs and their analogues in body fluids by gas chromatography with surface ionization detection

Eleven diphenylmethane antihistaminic drugs and their analogues were tested for their detection by capillary gas chromatography (GC) with surface ionization detection (SID). The GC—SID response was highest for doxylamine, diphenhydramine and orphenadrine and lowest for terodiline, clemastine and pipethanate. The detection limits for drugs with the highest response were 2–5 pg (ca. 6–20 fmol) on-column (100–250 pg/ml of body fluid). The detection limits with GC—SID were 10–100 times higher than those with GC with nitrogen—phosphorus detection. A detailed procedure for the isolation of the antihistaminics from human whole blood and urine by the use of Sep-Pak C₁₈ cartridges, prior to GC—SID, is also presented. The recoveries of the drugs (50 or 500 pmol), which had been added to 1 ml of body fluids, were>60%. The baselines remained steady as the column temperature was increased and the background was clean, especially for whole blood extracts.     

Porous graphitic carbon chromatography-tandem mass spectrometry for the study of isoprostanes in human cerebrospinal fluid

F2-isoprostanes are produced by the non-enzymatic peroxidation of arachidonic acid in membrane phospholipids. This paper describes a new method for the determination of all four classes of F2-isoprostanes in human cerebrospinal fluid (CSF) involving separation on a 1 mm x 150 mm porous graphitic carbon (PGC) column and detection by triple quadrupole mass spectrometry in negative-ion electrospray mode. The sample pre-treatment consisted of an ultrafiltration step, following which 300 microl of CSF sample could be injected directly onto a 1 mm x 10 mm PGC guard column functioning as a trap for the analytes. The loading solvent was Milli-Q water at 125 microl/min. After 3 min, the sample was switched into the separation column. The F2-isoprostanes were separated in 20 min using a linear solvent gradient comprising water, methanol, acetonitrile and ammonium hydroxide at a pH of 9.5 and a flow of 50 microl/min The limit of detection (calculated as 3S/N) was approximately 40 pM (14 pg/ml). The assay was linear within the examined range (18-450 pg/ml), using CSF spiked with iPF2alpha-III standard (r(2)>0.995). Repeatability data were calculated for CSF spiked to 90 pg/ml and the relative standard deviation (RSD) obtained was 3% (n=6).     

Determination of drugs of abuse and their metabolites in human plasma by liquid chromatography-mass spectrometry. An application to 156 road fatalities

A method, using 0.2 ml of plasma, was designed for the simultaneous determination of morphine, 6-monoacetylmorphine, amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB, benzoylecgonine and cocaine. The drugs were analysed by LC-MS, after solid phase extraction in the presence of the deuterated analogues. Reversed phase separation on an Atlantis dC18 column was achieved in 10 min, under gradient conditions. The method was full validated, including linearity (2-250 ng/ml, r2>0.99), recovery (>50%), within-day and between-day precision and accuracy (CV and bias <15%), limit of detection (0.5 and 1 ng/ml) and quantitation (2 ng/ml), relative ion intensities and no matrix effect was observed. The procedure showed to be sensitive and specific, and was applied to 156 real cases from road fatalities (7.1% cases positive to cocaine and 0.6% to designer drugs).     

Wet-chemical postcolumn reaction and fluorescence detection analysis of the reference interval of endogenous serum vitamin K1(20)

The reference interval for serum vitamin K1(20) levels was assayed in healthy fasting adults by a method based on high-performance liquid chromatography (HPLC). The isolation procedure involves a solvent extraction of the plasma lipids followed by two chromatographic steps, consisting of a purification of the extract on a semipreparative adsorption column and a final quantitation on a reverse-phase column. Vitamin K1(20) and vitamin K1(25), the internal standard, are monitored by fluorescence detection after postcolumn reduction with a methanolic solution of tetramethylammonium octahydridotriborate. This reaction is performed in an open tubular reaction coil at elevated temperature. The median plasma concentration in 50 healthy fasting adults was 247 pg/ml. The levels showed a skewed distribution with a range of 62 to 980 pg/ml [log x +/- 2 SD (log x)]. The method is linear over the entire physiological range and has a within-run precision of 3.6% (n = 5, mean = 311 pg/ml). The minimum detectable amount in serum is 50 pg/ml. Other extraction procedures resulted in lower recoveries or in interferences in the final measurement. The vitamin K1(20) levels as reported by other research groups are also discussed.     

Evaluation of reversed-phase columns for the analysis of heterocyclic aromatic amines by liquid chromatography-electrospray mass spectrometry

Liquid chromatography coupled to mass spectrometry (LC-MS), especially by the use of electrospray ionisation source (ESI), is currently used for the analysis of heterocyclic aromatic amines (HAs) in complex samples. The present paper describes the study of the performance of different narrow-bore reversed-phase columns to achieve the best chromatographic separation for the determination of 16 HAs by LC-ESI-MS in food samples. Different parameters such as peak symmetry, resolution and number of theoretical plates have been evaluated for each column, using different chromatographic conditions. The column that provided the best results was TSK Gel Semi-Micro ODS-80TS of Tosohaas. Quality parameters have been established, obtaining good short-term precision in all cases (relative standard deviation (R.S.D.) lower than 7.7%) and low limits of detection (<13 pg injected in MS and <16 pg injected in MS/MS). The content of HAs in two beef extracts have been determined.     

Detection of a mixed-backbone oligonucleotide (GEM 231) in liver and tumor tissues by capillary electrophoresis

A simple, rapid and sensitive method has been developed and validated for the analysis of a mixed-backbone oligonucleotide (GEM 231) in tumor tissues. The analysis was performed using a capillary electrophoresis (CE) system with UV detection. An extended light path (bubble cell) capillary column of 64.5 cm (effective length 56 cm)×50 μm I.D. is used as the separation column. The optimized chromatographic conditions were background electrolyte: sodium borate buffer (60 mM, pH 9.1), electrokinetic injection: 10 s, applied voltage: 30 kV, detection at λ=210 nm. A linear relationship was observed between the peak area and the amount of GEM 231 in the range of 1.0–1000 μg/ml. The lower detection limit of the drug was 100 pg with an average recovery of about 75±5%. The inter-day and intra-day relative standard deviations were <10%. Assay validation studies revealed that CE method is reproducible and specific for the determination of GEM 231 in tissue homogenates with a run time of less than 5 min.     

Determination of propiomazine in rat plasma by direct injection on coupled liquid chromatography columns with electrochemical detection

This method describes the determination of propiomazine by direct injection of rat plasma into a chromatography system based on coupled reversed-phase columns. An extraction column, packed with porous silica particles with covalent-bound ₁-acid glycoprotein (AGP), was used to separate the plasma proteins from the analyte. After isolation the analyte was transferred to the analytical column for separation and detection. Propiomazine was detected by an electrochemical detector and the limit of quantification was 2.0 ng/ml (100 pg injected). The absolute recovery was 80.9±2.4% at 9.0 ng/ml level. The inter-day and intra-day precision was 10.9% (5.6 ng/ml) and 2.8% (9.0 ng/ml), respectively.     

Determination of cocaine, norcocaine, benzoylecgonine and ecgonine methyl ester in rat plasma by high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatographic method with ultraviolet detection at 235 nm is described for the determination of cocaine and its metabolites benzoylecgonine, norcocaine and ecgonine methyl ester in rat plasma, collected during toxicity studies. Following simultaneous solid-phase extraction of all analytes and the internal standard tropacocaine, cocaine, benzoylecgonine and norcocaine were separated on a C₁₈ column. Ecgonine methyl ester and cocaine were separated on coupled cyanopropyl and silica columns, following derivatization of ecgonine methyl ester to p-fluorococaine. The extraction efficiencies of these compounds from plasma ranged from 78 to 87%, while the limits of detection ranged from 35 to 90 ng/ml. The assay was linear from 300 to 5000 ng/ml, and the within-day precision 2 to 8% over this concentration range.     

Direct detection and quantification of 19-norandrosterone sulfate in human urine by liquid chromatography-linear ion trap mass spectrometry

19-Norandrosterone sulfate (19-NAS) is the sulfoconjugated form of 19-norandrosterone (19-NA), the major metabolite of the steroid nandrolone. A sensitive and accurate liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the direct measurement of 19-NAS in human urine samples. The method involved a quaternary amine SPE protocol and subsequently injection of the extract onto an analytical column (Uptisphere ODB, 150 mm x 3.0 mm, 5 microm) for chromatographic separation and mass spectrometry detection in negative electrospray ionisation mode. The sulfoconjugate of 19-NA was identified in urine by comparison of mass spectra and retention time with a reference substance. The limit of detection (LOD) and lowest limit of quantification (LLOQ) of 19-NAS were of 40 pg/mL and 200 pg/mL, respectively. For a nominal concentration of 2 ng/mL, recovery (94%), intra-day precision (2.7%), intra-assay precision (6.6%) and inter-assay precision (14.3%) were determined. Finally, this analytical method was applied for quantifying the concentration of 19-NAS in doping samples, using calibration curves (0.2-20 ng/mL) and the standard-addition method. The results show the feasibility of applying this LC-MS/MS assay as a complementary tool to detect misuse of nandrolone or nandrolone precursors.     

Rapid diagnosis of phenylketonuria and other aminoacidemias by quantitative analysis of amino acids in neonatal blood spots by gas chromatography-mass spectrometry

A reversed-phase HPLC method compatible with evaporative light scattering (ELS) and electrospray mass spectrometric (ES-MS) detection was developed for separation of phosphatidylserine (PS) molecular species. The method was optimised for separation of three disaturated synthetic species: dipalmitoyl glycerophosphoserine, palmitoyl-stearoyl glycerophosphoserine and distearoyl glycerophosphoserine using isocratic elution with a mixture of 2-propanol, tetrahydrofuran and ammonium formate. Baseline separation was obtained on three different columns: one polystyrene/divinylbenzene (PS/DVB) column and two silica based C(18) and C(30) columns. The best chromatographic resolution was achieved with the C(30) column. The limit of detection for DPPS was 5 microg/ml (S/N=3) with ELS detection and 0.1 microg/ml (S/N=3) with negative ion ES-MS in the single ion monitoring mode. Baseline separation of the five main species in a biological PS sample, bovine brain PS, was obtained with the PS/DVB column. Species identification was done by using the retention times of the intact PS species and their corresponding carboxylate anion fragments obtained by in-source fragmentation. Data have shown that individual PS species can be identified by their retention times using direct ELS detection in a mixture of disaturated PS species. However, for the bovine brain PS electrospray-MS detection was necessary for species identification due to the many possible fatty acid combinations in biological PS.     

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

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