Development of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Simultaneous Determination of 40 Drugs of Abuse in Human Urine: Application to Real Cases.

Drugs of abuse are constantly evolving, while new synthetized substances are constantly emerging to avoid regulations. However, traditional drugs such as cocaine and amphetamine are still two of the most consumed drugs in the world. It is important, therefore, to provide suitable multiresidue methods for determining a wide range of drugs for use in toxicological and forensic analyses. The aim of this study is to develop a method for determining several families of drugs of abuse, including classic drugs, new psychoactive substances and some of their metabolites, in urine by liquid chromatography-tandem mass spectrometry. Urine is one of the most common biological matrices used in drug analysis because of its easy collection and a wide window of detection. In this study, we used solid-phase extraction to remove interferences and extract analytes from urine. Four different mixed-mode cation-exchange commercial sorbents were evaluated. The best results, in terms of apparent recoveries, were achieved with one of the strong cationic sorbents, ExtraBond SCX. The method achieved detection limits from 0.003 to 0.500 ng/mL and quantification limits from 0.050 to 1.500 ng/mL, which are suitable for determining these compounds at the usual levels found in the urine of drug users. The applicability of this method was demonstrated by analyzing real urine specimens from women following a detoxification program. Our results showed that the drug most consumed was cocaine, since it was detected in most urine specimens together with its main metabolite, benzoylecgonine. The polyconsumption of drugs from different families was also observed in some urine samples analyzed.

Comparison of different chiral selectors for the enantiomeric determination of amphetamine-type substances in human urine by solid-phase extraction followed by capillary electrophoresis-tandem mass spectrometry.

The present study develops a method for the enantioseparation of a group of amphetamines and their metabolites in urine by CE coupled to MS/MS (CE-MS/MS). Amphetamines present a chiral center and thus two enantiomers, which is important from a toxicological point of view because they may have different pharmacokinetic and pharmacological properties. It is therefore essential to find suitable methods to distinguish both enantiomers. Today the use of CE is becoming more important in this field since, with the simple addition of a chiral selector to the background electrolyte, the enantioseparation can easily be achieved. However, when CE is coupled to MS, the use of volatile chiral selectors and compatible background electrolytes or other strategies such as the countercurrent migration approach are required to avoid contamination of the ion source from nonvolatile species. In the present study, we use the latter strategy to evaluate six different chiral selectors using CE-MS/MS. As a sample pre-treatment, two cationic-exchange sorbents-Oasis WCX and Oasis MCX-are compared for the urine pre-treatment. Using this method, it was possible to achieve the complete chiral separation of the amphetamines under study with detection limits ranging between 0.8 and 1.5 ng/mL and method quantification limits between 2.0 and 8.0 ng/mL. Matrix-matched calibration curves up to 150 ng/mL were used to cover the usual concentration ranges at which amphetamines have generally been found in toxicological and forensic analyses.

Enantiodetermination of R,S-3,4-methylenedioxypyrovalerone in urine samples by high pressure in-line solid-phase extraction capillary electrophoresis-mass spectrometry.

This study presents for the first time an in-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) method for the enantiodetermination of drugs of abuse in urine samples. The enantioseparation of R,S-3,4-methylenedioxypyrovalerone (R,S-MDPV) was achieved with a 10 mM ammonium acetate BGE (pH 7) that contained 0.5% (m/v) of sulphated-α-CD as chiral selector. At these pH conditions, this CD was negatively charged, which prevented its entrance into the mass spectrometer since it migrates in the opposite direction. To improve sensitivity, an in-line SPE-CE-MS method using high pressure for sample introduction (i.e. 20 min at 3 bars) was developed. Furthermore, the conditioning procedure and the first part of the electrophoretic separation were performed by switching off the nebulizer gas and the ionization source voltage to avoid non-volatile contaminant arrival into the mass spectrometer. The developed methodology was validated by analyzing urine samples, which required a very simple liquid-liquid extraction (LLE) sample pretreatment. Linearity ranged from 30 to 250 ng mL-1, limit of detection (LOD) was 10 ng mL-1, relative standard deviation (RSD) values were below 10.5% in terms of intra-day and inter-day precision and the relative error values were below 9% for peak areas accuracy.

A Fast Analytical Method for Determining Synthetic Cathinones in Oral Fluid by Liquid Chromatography-Tandem Mass Spectrometry.

In this paper, we present a method for simultaneously determining 11 synthetic cathinones in oral fluid (OF) by liquid chromatography-tandem mass spectrometry. Synthetic cathinones, a wide variety of which are available on the market, are constantly evolving. It is therefore important to provide efficient methods for determining cathinones in different matrices. A common matrix for detecting recent drug intake is OF, which can easily be collected using one of numerous commercial devices. Most methods aimed at determining drugs in biological samples such as OF require labor-intensive and time-consuming sample-preparation steps. However, the pretreatment of complex samples is often a challenge in the development of a method. For this reason, in this paper, we present a simple, easy-to-handle alternative that uses a Salivette® device and pretreats the sample in the same device. Matrix-matched calibration curves were used to cover the concentration range at which these substances are usually present in the OF from drug consumers. The method detection limits ranged from 0.003 to 0.03 ng/g, and the method quantification limits were set at 0.075 ng/g. This is a simple, rapid and sensitive method with good potential for determining recent drug consumption in OF.

Field-amplified sample injection combined with CE for the enantiodetermination of cathinones in urine samples.

This work presents a capillary electrophoresis methodology for the enantiodetermination of cathinones in urine employing a liquid-liquid extraction sample pretreatment. The cathinones were enantioseparated by adding a mixture of 8 mM 2-hydroxypropyl ß-cyclodextrin and 5 mM ß-cyclodextrin to the background electrolyte, which consists of 70 mM of monosodium phosphate aqueous solution at pH 2.5. Field-amplified sample injection was used as preconcentration strategy to improve the sensitivity. We studied various parameters that affect this stacking strategy, in particular, the sample solvent and its pH, the presence or absence of a low conductivity solvent plug introduced before the sample injection, the nature and volume of this plug, and the voltage and time of the electrokinetic injection of the sample. The optimum conditions were achieved by injecting a plug of isopropanol:H2 O 50/50 at 50 mbar for 5 s prior to the electrokinetic injection of the sample prepared in an aqueous solution of HCl 10-6  M. The sensitivity enhancement factors were from 562 to 601 in terms of peak area and from 444 to 472 in terms of peak height. The method was validated by analyzing spiked urine samples, obtaining a linear range of 25 to 1000 ng/mL and limits of detection ranging from 15 to 45 ng/mL.

Enantioselective determination of cathinones in urine by high pressure in-line SPE-CE.

This work presents a strategy based on the in-line coupling of SPE and CE for the chiral determination of cathinones (R,S-mephedrone, R,S-4-methylephedrine, and R,S- methylenedioxypyrovalerone) in urine samples, using a sample pretreatment based on liquid-liquid extraction. The chiral separation of the compounds is achieved by adding a mixture of 8 mM 2-hydroxypropil ß-CD and 5 mM ß-CD to the BGE, which consists of 70 mM of monosodium phosphate aqueous solution at pH 2.5. Oasis HLB was the selected sorbent for the in-line SPE device, and to reduce analysis time and LODs, several parameters affecting the in-line SPE system were evaluated, such as pressure and time of sample injection and dimensions of the SPE device. The highest preconcentration factors were achieved by using 3 bar of injection pressure for 20 min with an in-line SPE device of 2 mm length and 150 µm of i.d. The developed method was applied to determine the presence of the compounds in spiked urine samples. The LODs obtained were between 3 and 8 ng/mL, and these levels were below the usual concentrations at which these drugs are present in urine from cathinone abusers. Thus, the optimized method has the potential to be applied for toxicological and forensic purposes.

Sensitivity Enhancement in Capillary Electrophoresis Using Magnetic Particles as Solid-Phase Extraction Sorbents for the Determination of Drugs of Abuse in Urine.

Over the last few years, different types of magnetic particles have been investigated and successfully used in sample preparation, of which iron oxides are the most popular, due to their low price and low toxicity. For analytical purposes, these particles have always been modified and functionalized with different materials to improve their stability and introduce new surface properties. Here we describe the preparation of silica-coated iron oxide particles functionalized with C18 and their application as solid-phase extraction sorbents coupled in-line with capillary electrophoresis for determining drugs of abuse in human urine.

Findings in the hair of drug abusers using pressurized liquid extraction and solid-phase extraction coupled in-line with capillary electrophoresis.

A suitable method has been developed and validated for simultaneously determining cocaine and its major metabolite, benzoylecgonine, 6-acetylmorphine, codeine, morphine and the methadone enantiomers in human hair samples by the in-line coupling between SPE and CyD-assisted CE with a previous sample pretreatment procedure based on pressurized liquid extraction. Optimal separation was achieved on a fused silica-capillary of 50µm i.d. and 80cm total length using 11mM α-CyD in an aqueous solution of 80mM sodium phosphate at pH 2.5 as the separation medium and an applied voltage of 30kV. The SPE-CE device consisted of a short length of capillary packed with Oasis HLB sorbent, which was inserted near to the inlet end of the CE capillary. Several parameters affecting the in-line preconcentration were evaluated. The LOQs reached for hair samples were in the range of 0.3-2.5ng/mg with satisfactory analytical precision in both intraday and day-to-day experiments (RSDs <13%). Relative recoveries greater than 80% were obtained. The method has successfully been applied to the determination of these drugs of abuse in segmented hair from drug abusers who were undergoing methadone maintenance treatment. The results were consistent with the patients' statements, indicating that the method established herein can be used for verifying a history of drug abuse.

Enantioselective determination of cathinone derivatives in human hair by capillary electrophoresis combined in-line with solid-phase extraction.

A suitable method has been developed and validated for the chiral separation and determination of R,S-mephedrone and one of its metabolites, R,S-4-methylephedrine, and R,S-methylenedioxypyrovalerone (R,S-MDPV) in human hair samples by the in-line coupling between SPE and CD-assisted CE with a previous sample pretreatment procedure based on pressurized liquid extraction. Optimum separation was achieved on a fused silica-capillary of 50 µm id and 80 cm total length using 12 mg/mL ß-CD in an aqueous solution of 80 mM disodium phosphate at pH 2.5 as the BGE and an applied voltage of 30 kV. The SPE-CE device consists of a short length of a capillary of 2 mm packed with Oasis HLB sorbent, which was inserted near to the inlet end of the CE capillary. Several parameters affecting the in-line preconcentration were evaluated. The LOQs reached for hair samples were 0.05 ng/mg for the enantiomers of mephedrone and its metabolite, and 0.40 ng/mg for the enantiomers of MDPV. The RSDs (%) obtained in intra- and interday studies were less than 10% and the relative recoveries were greater than 80%. The method established in this paper is advantageous for its simplicity, overall analysis time and ability to provide information of both enantiomers of a chiral drug in hair samples.

Capillary electrophoresis combined in-line with solid-phase extraction using magnetic particles as new adsorbents for the determination of drugs of abuse in human urine.

A simple approach is presented based on the in-line coupling between magnetic particles-based SPE and CE. Silica-coated iron oxide particles functionalized with C18 were successfully synthesized and used as a reverse-phase sorbent for in-line SPE-CE. Magnets were used to locally immobilize these sorbents inside the capillary. Four drugs of abuse were preconcentrated and determined in urine samples using the developed method with a simple pretreatment procedure based on LLE. Several parameters affecting the preconcentration were evaluated. The obtained results show that this strategy enhanced detection sensitivity in the range of 125-700-fold compared with CE without preconcentration. The developed method provides LODs (S/N = 3) for standard samples in the range of 0.5-20 ng/mL with satisfactory analytical precision, in both intraday and day-to-day experiments (RSDs <20%). The LODs (S/N = 3) reached for urine samples were in the range of 20-50 ng/mL. Relative recoveries greater than 75.9% were obtained. The established method has been applied to the analysis of drugs of abuse in urine samples from drug abusers.

Single-drop microextraction combined in-line with capillary electrophoresis for the determination of nonsteroidal anti-inflammatory drugs in urine samples.

This study describes a method to determine nonsteroidal anti-inflammatory drugs (NSAIDs) in urine samples based on the use of single-drop microextraction (SDME) in a three-phase design as a preconcentration technique coupled in-line to capillary electrophoresis. Different parameters affecting the extraction efficiency of the SDME process were evaluated (e.g. type of extractant, volume of the microdroplet, and extraction time). The developed method was successfully applied to the analysis of human urine samples with LODs ranging between 1.0 and 2.5 µg/mL for all of the NSAIDs under study. This method shows RSD values ranging from 8.5 to 15.3% in interday analysis. The enrichment factors were calculated, resulting 27-fold for ketoprofen, 14-fold for diclofenac, 12-fold for ibuprofen, and 44-fold naproxen. Samples were analyzed applying the SDME-CE method and the obtained results presented satisfactory recovery values (82-115%). The overall method can be considered a promising approach for the analysis of NSAIDs in urine samples after minimal sample pretreatment.

Determination of cocaine in abuser hairs by CE: monitoring compliance to a detoxification program.

BACKGROUND: Segmental hair analysis was performed to verify the cocaine withdrawal and compliance to the therapy of four cocaine abusers that were following a drug detoxification program. RESULTS/METHODOLOGY: Cocaine and its major metabolite, benzoylecgonine, were preconcentrated and determined by in-line SPE and CE with prior isolation of the analytes from the hair matrix by an overnight acidic incubation procedure. The LODs obtained for hair samples were 0.02 ng/mg for cocaine and 0.1 ng/mg for benzoylecgonine. CONCLUSION: Our results showed that the established method, in conjunction with a segmental hair analysis, is suitable for determining drug abuse histories, being very useful in forensic toxicological laboratories as well as in rehabilitation and addiction treatment programs.

In-line solid-phase extraction-capillary zone electrophoresis for the determination of barbiturate drugs in human urine.

The abuse of barbiturate drugs is widespread, and the development of methods for their efficient separation and quantification is needed. Three barbiturate drugs were preconcentrated and determined by in-line solid-phase extraction (SPE) capillary electrophoresis (CE) in urine samples. Different parameters affecting preconcentration were evaluated, such as the sample pH, the volume of the elution plug and the sample injection time. This strategy enhanced the detection sensitivity in the range of 170- to 1840-fold, compared with normal hydrodynamic injection. The method provides limits of detection (LODs) for standard samples in the range of 0.5 to 5 ng/mL with good repeatability and reproducibility values. The LODs obtained for urine samples were in the range of 5 to 60 ng/mL. The validation with human urine samples spiked with the studied compounds demonstrated the applicability of the optimized method. This method provides a reproducible and sensitive analysis of urine samples in the determination of barbiturates drugs.

Electrokinetic supercharging in CE for the separation and preconcentration of barbiturate drugs in urine samples.

Three barbiturate drugs, barbital, phenobarbital, and secobarbital were separated and analyzed by electrokinetic supercharging. The influence of different parameters on electrokinetic supercharging performance was evaluated using both univariated and multivariated optimization processes. The parameters studied were sample pH, concentration, and length of the leading and terminating electrolytes, electrokinetic injection of the sample and composition and hydrodynamic injection of the solvent plug. The leading electrolyte (50 mM NaCl) was hydrodynamically injected (50 mbar × 120 s) prior to the sample that was adjusted to pH 9.6 and electrokinetically injected at -8.5 kV for 300 s. The terminating electrolyte (100 mM of 2-(cyclohexylamino) ethanesulphonic acid) was then hydrodynamically injected (50 mbar × 140 s). The results showed that this strategy enhanced detection sensitivity around 1050-fold compared with normal hydrodynamic injection, providing detection limits ranging between 1.5 and 2.1 ng/mL for standard samples with good repeatability in terms of peak area (values of relative standard deviation, %RSD < 3). The applicability of the optimized method was demonstrated by the analysis of human urine samples spiked with the studied compounds at different concentration levels and further liquid-liquid extraction step. The estimated detection limits obtained in the urine samples extract ranged between 8 and 15 ng/mL.

Different strategies for the preconcentration and separation of parabens by capillary electrophoresis.

Several strategies, namely, large volume sample stacking (LVSS), field-amplified sample injection (FASI), sweeping, and in-line SPE-CE, were investigated for the simultaneous separation and preconcentration of a group of parabens. A BGE consisting of 20 mM sodium dihydrogenphosphate (pH 2.28) and 150 mM SDS with 15% ACN was used for the separation and preconcentration of the compounds by sweeping, and a BGE consisting of 30 mM sodium borate (pH 9.5) was used for the separation and preconcentration of the compounds by LVSS, FASI, and in-line SPE-CE. Several factors affecting the preconcentration process were investigated in order to obtain the maximum enhancement of sensitivity. The LODs obtained for parabens were in the range of 18-27, 3-4, 2, and 0.01-0.02 ng/mL, and the sensitivity evaluated in terms of LODs was improved up to 29-, 77-, 120-, and 18,400-fold for sweeping, LVSS, FASI, and in-line SPE-CE, respectively. These preconcentration techniques showed potential as good strategies for focusing parabens. The four methods were validated with standard samples to show the potential of these techniques for future applications in real samples, such as biological and environmental samples.

Determination of UV filters in river water samples by in-line SPE-CE-MS.

The use of SPE coupled in-line to CE using electrospray MS detection (in-line SPE-CE-ESI-MS) was investigated for the preconcentration and separation of four UV filters: benzophenone-3, 2,2-dihydroxy-4-methoxybenzophenone, 2,4-dihydroxybenzophenone and 2-phenylbenzimidazole-5-sulphonic acid. First, a CE-ESI-MS method was developed and validated using standard samples, obtaining LODs between 0.06 µg/mL and 0.40 µg/mL. For the in-line SPE-CE-ESI-MS method, three different sorbents were evaluated and compared: Oasis HLB, Oasis MCX, and Oasis MAX. For each sorbent, the main parameters affecting the preconcentration performance, such as sample pH, volume, and composition of the elution plug, and sample injection time were studied. The Oasis MCX sorbent showed the best performance and was used to validate the in-line SPE-CE-ESI-MS methodology. The LODs reached for standard samples were in the range between 0.01 and 0.05 ng/mL with good reproducibility and the developed strategy provided sensitivity enhancement factors between 3400-fold and 34 000-fold. The applicability of the developed methodology was demonstrated by the analysis of UV filters in river water samples.

In-line solid-phase extraction-capillary electrophoresis coupled with mass spectrometry for determination of drugs of abuse in human urine.

In-line solid-phase extraction-capillary electrophoresis coupled with mass spectrometric detection (SPE-CE-MS) has been used for determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine (COD), hydrocodeine (HCOD), and 6-acetylmorphine (6AM) in urine. The preconcentration system consists of a small capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoresis capillary. The SPE-CE-MS experimental conditions were optimized as follows: the sample (adjusted to pH 6.0) was loaded at 930 mbar for 60 min, elution was performed with methanol at 50 mbar for 35 s, 60 mmol L(-1) ammonium acetate at pH 3.8 was used as running buffer, the separation voltage was 30 kV, and the sheath liquid at a flow rate of 5.0 µL min(-1) was isopropanol-water 50:50 (v/v) containing 0.5% acetic acid. Analysis of urine samples spiked with the four drugs and diluted 1:1 (v/v) was studied in the linear range 0.08-10 ng mL(-1). Detection limits (LODs) (S/N = 3) were between 0.013 and 0.210 ng mL(-1). Repeatability (expressed as relative standard deviation) was below 7.2%. The method developed enables simple and effective determination of these drugs of abuse in urine samples at the levels encountered in toxicology and doping.

Investigation of in-line solid-phase extraction capillary electrophoresis for the analysis of drugs of abuse and their metabolites in water samples.

In this study, in-line solid-phase extraction (SPE) was used as an enrichment technique in combination with CE for the preconcentration and separation of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine (COC), codeine (COD) and 6-acetylmorphine (6AM). The separation buffer (BGE) used was 80 mM disodium phosphate anhydrous and 6 mM of HCl (final BGE pH of 3). The SPE extractor consists of a small segment of capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoretic capillary. Different parameters affecting preconcentration were evaluated, such as sample pH, the volume of the elution plug and sample injection time. The detection limits (LODs) reached for standard samples by in-line SPE-CE-UV ranged between 50 and 200 ng/L, with sensitivity enhancement factors ranging from 2300 to 5300. Reproducibility values (expressed in terms of relative standard deviation) were below 7.6% for standard samples. This is a simple and an effective method for the determination of the studied drugs of abuse and their metabolites. The applicability of the developed method was demonstrated in tap and river water samples which were directly analyzed without any off-line pretreatment. Analytical parameters were evaluated and LODs were between 70 and 270 ng/L with relative recoveries between 85 and 97%.

An in-line SPE strategy to enhance sensitivity in CE for the determination of pharmaceutical compounds in river water samples.

In this study, the suitability of solid-phase extraction (SPE) coupled in-line to CE with UV-Vis detection was evaluated for the preconcentration and separation of diluted solutions of five pharmaceuticals compounds: benzafibrate, piroxicam, diclofenac sodium, naproxen and clofibric acid. An SPE analyte concentrator containing Oasis(®) HLB sorbent was constructed without frits and placed near the inlet end of the separation capillary. Different parameters such as sample pH, composition and volume of the elution plug and sample loading time were studied in order to obtain the maximum preconcentration factors. The LODs reached for standard samples were in the range 0.06-0.5 ng/mL with good reproducibility, and the developed strategy provides sensitivity enhancement factors around 14,000-fold in peak area and 5900-fold in peak height compared with the normal hydrodynamic injection. Finally, river water samples fortified with the pharmaceutical compounds were analyzed by the developed in-line SPE-CE-UV method in order to show the potential of the methodology for the analysis of environmental aquatic samples. For these samples, high values of relative recoveries, between 73-107% and 79-103% for two concentration levels, 5 and 25 ng/mL, respectively, were obtained and LODs ranged between 0.19 and 1 ng/mL.

Simultaneous determination of weakly ionizable analytes in urine and plasma samples by transient pseudo-isotachophoresis in capillary zone electrophoresis.

A rapid method for the simultaneous determination of several non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma and urine was developed using transient pseudo-isotachophoresis (ITP) in capillary zone electrophoresis (CZE). The influence of different parameters on resolution and preconcentration efficiency, such as background electrolyte (BGE) composition, sample injection, sample matrix composition, and pH, were studied to optimize the transient pseudo-ITP performance. Optimized conditions were a BGE consisting of 100 mM Na(2)B(4)O(7) in 10% aqueous MeOH solution and hydrodynamic injection of the sample at 50 mbar for 90 s. The sample was prepared in a solution mixture of 1% NaCl/ethanol (30:70 v/v) at pH 10. Our results show that this simple strategy offers improved sensitivity compared to conventional CZE analysis, reaching a 45-fold preconcentration factor. The detection limits (LODs) were as low as 0.07 mg/L for standard samples with good repeatability (values of relative standard deviation, %RSD < 11%). The method was applied to the analysis of NSAIDs in biological samples. Validation for human plasma and urine samples demonstrated good linearity, low detection limits, and satisfactory repeatability values.