Single drop liquid-liquid-liquid microextraction of methamphetamine and amphetamine in urine

Single drop liquid-liquid-liquid microextraction (LLLME) combined with high performance liquid chromatography (HPLC)-UV detection was investigated for the determination of a popular drug of abuse, methamphetamine (MAP), and its major metabolite, amphetamine (AP), in urine samples. The target compounds were extracted from NaOH modified sample solution to a thin layer of organic solvent membrane, and back-extracted to an acidic acceptor drop suspended on the tip of a 50-microL HPLC syringe in the aforementioned organic layer. This syringe was also used for direct injection after extraction. Factors affecting extraction efficiency were studied. At optimal conditions, the overall enrichment factor (EF) was 500-fold for AP and 730-fold for MAP, respectively. The method exhibited a wide linear range (1.0-1500 microg/L), low detection limit (0.5 microg/L), and good repeatability (RSD<5.0%) for both analytes. The feasibility of the method was demonstrated by the analysis of human urine samples.     

Headspace liquid-phase microextraction of methamphetamine and amphetamine in urine by an aqueous drop

This study developed a headspace liquid-phase microextraction (LPME) method by using a single aqueous drop in combination with high performance liquid chromatography (HPLC)-UV detection for the determination of methamphetamine (MAP) and amphetamine (AP) in urine samples. The analytes, volatile and basic, were released from sample matrix into the headspace first, and then protonated and dissolved in an aqueous H₃PO₄ drop hanging in the headspace by a HPLC syringe. After extraction, this drop was directly injected into HPLC. Parameters affecting extraction efficiency were investigated and optimized. This method showed good linearity in the investigated concentration range of 1.0-1500 μg L⁻¹, repeatability of the extraction (R.S.D. < 5%, n = 6), and low detection limits (0.3 μg L⁻¹ for both analytes). Enrichment factors of about 400-fold and 220-fold were achieved for MAP and AP, respectively, at optimum conditions. The feasibility of the method was demonstrated by analyzing human urine samples.     

Distribution characteristics of methamphetamine and amphetamine in urine and hair specimens collected from alleged methamphetamine users in northern Taiwan

This study was conducted to better understand the distribution characteristics of methamphetamine and amphetamine in urine and hair specimens collected from alleged methamphetamine users in the local population. It is anticipated that the data hereby obtained will be helpful to the interpretation of the time and pattern of drug use. Eight alleged methamphetamine-using arrestees from Keelung Police Department (north of Taipei, Taiwan) consented to contribute both urine and hair specimens. Each arrestee contributed seven urine specimens collected at 0, 12, 24, 48, 72, 96, and 120 h, respectively, after the arrest. Hair specimens were cut into 2-cm sections. The limits of detection and quantitation of the urine protocol were 40 and 50 ng/mL, respectively, for both amphetamine and methamphetamine, while the corresponding limits of detection and quantitation for the hair protocol were 0.8 and 1.0 ng/mg, respectively. The concentration variations of methamphetamine and amphetamine in the urine specimens exhibited three distinct patterns: (a) continuous decrease in the analytes’ concentrations for specimens collected at hours 0-120; (b) increase in the analytes’ concentrations in specimens collected at hours 0-12, followed by decrease; (c) increase in analytes’ concentrations in specimens collected at later times. Together with the amphetamine/methamphetamine concentration ratios found in these urine specimens, the observed trends in the changes of the analytes’ concentrations are helpful for the interpretation on the time of drug use. Unlike urine specimens, amphetamine/methamphetamine concentration ratios in various hair specimens and hair sections remain relatively constant.     

HPLC with fluorescence detection of methamphetamine and amphetamine in segmentally analyzed human hair

A sensitive high-performance liquid chromatographic method with fluorescence detection for determining methamphetamine and its major metabolite, amphetamine, in abusers' hair segments was developed. Methamphetamine and amphetamine in hair samples collected from addicts were extracted into acidified methanol, derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride, separated isocratically on an ODS column using TRIS-HCl buffer (0.1 mol dm-3, pH 7.0)-methanol (30 + 70 v/v) as the mobile phase and the derivatives were detected fluorimetrically at 440 nm (lambda ex 330 nm). Calibration curves obtained by using control human hair spiked with standard solutions were linear (r > or = 0.999) up to at least 676.1 ng mg-1 for amphetamine and 746.1 ng mg-1 for methamphetamine. The detection limits at a signal-to-noise ratio of 3 were 51.4 and 74.6 pg mg-1 hair for amphetamine and methamphetamine, respectively. Using control hair spiked with standard solutions, the intra- and inter-day relative standard deviations (n = 5) were < or = 8.6% for both the target compounds. The method was successfully applied to the segmental analyses of methamphetamine abusers' hair samples.     

Polarization fluoroimmunoassays for the specific determination of amphetamine and methamphetamine in urine with the abbott TDx-analyzer

Two polarization fluoroimmunoassays for the screening of amphetamine and methamphetamine in urine were adapted for use with the Abbott TD_x-Analyzer running in the automatic mode. The detection limits of amphetamine and methamphetamine in 10 μl of urine were 0.03 and 0.05 mg l^?1, respectively. These assays were specific for the determination of amphetamine (cross-reactivity with methamphetamine 1%) or for the determination of methamphetamine (cross-reactivity with amphetamine 2.5%). No significant cross-reaction was observed with other drugs of abuse (ephedrine, morphine and butabarbital).     

Determination of amphetamine and methamphetamine in urine by solid phase extraction and ion-pair liquid chromatography-electrospray-tandem mass spectrometry

A method using a solid phase extraction (SPE) and ion-pair liquid chromatography-electrospray-tandem mass spectrometry (LC-ES-MS/MS) was developed for determination of amphetamine (Amp) and methamphetamine (mAmp) in urine samples. A reversed phase C₁₈ column was utilized for LC separation and MS/MS was used for detection. Trifluoroacetic acid was added to the mobile phase as an ion-pairing reagent. MS² was employed for quantitative determination. In addition, d₈-amphetamine and d₈-methamphetamine were used as internal standards. An Oasis HLB SPE cartridge, which has hydrophilic and lipophilic functions, was utilized for sample pre-treatment. Recoveries ranging from 97.3 to 102.1% were measured. Good linear ranges, 5-500 ng/ml, for Amp and mAmp were determined. The detection limit of each analytical compound, based on a signal-to-noise ratio of 3, was approximately 1 ng/ml. The applicability of this newly developed method was examined by analyzing several urine samples from drug users.     

Rapid and highly sensitive method for determination of methamphetamine and amphetamine in urine by electron-capture gas chromatography

A rapid and sensitive method for determination of methamphetamine and amphetamine in urine was developed by using electron-capture gas chromatography. The extraction procedure, the experimental conditions for pentafluorobenzoyl derivative formation and the percentage recovery of the drugs from urine are described. The pentafluorobenzoyl derivative of methamphetamine showed a higher electron-capture sensitivity and was detected in at least 23-fold lower concentration than the heptafluorobutyryl derivative which is commonly used as a derivatizing agent for the amine. The detection limit of pentafluorobenzoyl derivatives of methamphetamine and amphetamine was ca. 10 pg. A concentration as low as 10 ng/ml of methamphetamine and amphetamine in urine was easily detected by this method.     

Rapid screening and confirmation of amphetamine, methamphetamine, methadone, and methadone metabolite in urine by gas/thin layer chromatography.

A method suitable for large scale screening and confirmation of urine speciments for amphetamine, methamphetamine, methadone, and its primary metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenlypyrrolidine) is described. The drugs are extracted from alkaline urine into an organic solvent. The amphetamine drugs are then back-extracted into a small volume of acid and identified by gas chromatography both as free bases on a 10% Apiezon L-10% KOH column and as their trifluoracetamide derivatives on a 3% OV-17 column. The organic layer, which still contains methadone and its primary metabolite, is analyzed by split-sample thin-layer chromatography using two solvent systems: ethyl acetate: methylene cloride: concentrated ammonium hydroxide (90:10:0.7) and methanol: chloroform: concentrated ammonium hydroxide (74:25:0.8). These solvent systems separate methadone from its primary metabolite without interference from other drugs or urinary substances.     

Incorporation of methamphetamine and amphetamine in human hair following controlled oral methamphetamine administration

Background

Although hair testing is well established for the assessment of past drug exposure, uncertainties persist about mechanisms of drug incorporation into hair and interpretation of results. The aim of this study was to administer methamphetamine (MAMP) under controlled conditions as a model drug to investigate drug incorporation into human hair.

Material and methods

Seven volunteers with a history of stimulant use received 4 × 10 mg (low) doses of sustained release S-(+)-MAMP HCl within 1 week, with weekly head hair samples collected by shaving. 3 weeks later, 4 of them received 4 × 20 mg (high) doses. After extensive isopropanol/phosphate buffer washing of the hair, MAMP and its metabolite amphetamine (AMP) concentrations were determined in all weekly hair samples by LC–MS–MS in selected reaction monitoring mode with the undeca- and deca-deuterated drugs, respectively, as internal standards (LLOQ, 0.005 ng mg⁻¹).

Results

MAMP T_max occurred from 1 to 2 weeks after both doses, with C_max ranging from 0.6 to 3.5 ng mg⁻¹ after the low and 1.2 to 5.3 ng mg⁻¹ after the high MAMP doses. AMP C_max in hair was 0.1–0.3 ng mg⁻¹ and 0.2–0.5 ng mg⁻¹, respectively, for low and high doses. Highly dose-related concentrations within subjects, but large variability between subjects were observed. MAMP concentrations were above the 0.2 ng mg⁻¹ cut-off for at least 2 weeks following administration of both low and high doses. The overall AMP/MAMP ratio ranged from 0.07 to 0.37 with a mean value of 0.15 ± 0.07, and a median of 0.13. The percentage of MAMP and AMP removed with the washing procedure decreased with time after administration. A strong correlation was found between area under the curve of MAMP (r² = 0.90, p = 0.00) and AMP (r² = 0.94, p = 0.00) concentrations calculated for the 3-week period following administration and the total melanin concentration in hair. Significant correlations were observed also between C_max and melanin.

Conclusions

This study demonstrated that despite large inter-individual differences, the incorporation of MAMP and AMP into hair is dose-related with much of the observed scatter of MAMP and AMP concentrations explained by melanin concentration in hair.     

Chiral separation of 3,4-methylenedioxymeth- amphetamine and related compounds in clandestine tablets and urine samples by capillary electrophoresis/fluorescence spectroscopy

The R-(-)- and S-(+)-isomers of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolite 3,4-methylenedioxyamphetamine (MDA) were prepared, identified by gas chromatography/mass spectrometry (GC/MS) and then used as standards in a series of capillary electrophoresis (CE) experiments. Using these R-(-)- and S-(+)-isomers, the distribution of (RS)-MDA and (RS)-MDMA stereoisomers in clandestine tablets and suspect urine samples were identified. Several electrophoretic parameters, such as the concentration of beta-cyclodextrin used in the electrophoretic separation and the amount of organic solvents required for the separation, were optimized.     

Mass spectra of bithiophthalide and related compounds

High resolution mass spectra of biphlhalidu ( 1 ), bithiophthalide ( 2 ), and thioindigo ( 3 ) were investigated and the major fragmentation patterns were elucidated. The simultaneous loss of two sulfur atoms from the molecular ion of 2 was particularly noteworthy.     

Determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxymethamphetamine in urine by online solid-phase extraction and ion-pairing liquid chromatography with detection by electrospray tandem mass spectrometry

A method using an online solid-phase extraction (SPE) and ion-pairing liquid chromatography with electrospray tandem mass spectrometry (LC/ES-MS/MS) was developed for determination of amphetamine (Amp), methamphetamine (mAmp), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples. A SPE cartridge column with both hydrophilic and lipophilic functions was utilized for online extraction. A reversed-phase C18 LC column was employed for LC separation and MS/MS was used for detection. Trifluoroacetic acid was added to the mobile phase as an ion-pairing reagent. This method was fully automated and the extraction and analysis procedures were controlled by a six-port switch valve. Recoveries ranging from 85-101% were measured. Good linear ranges (10-500 ng/mL) for Amp and mAmp were determined. For MDA, MDMA and MDEA, dual linear ranges were obtained from 5-100 and 100-500 ng/mL, respectively. The detection limit of each analytical compound, based on a signal-to-noise ratio of 3, ranged from 1-3 ng/mL. The applicability of this newly developed method was examined by analyzing several urine samples from drug users. Good agreement was obtained between the results from this method and a literature GC/MS method.     

Mass spectrometry of antibiotic kikumycin A,B and related compounds

The structures of kikumycin A and B, antiviral amidine antibiotics, have been elucidated by means of low and high resolution mass spectrometry. Amino acid composition and their sequences were also clearly demonstrated by this technique. The evidence for the fragmentation of terminal amidines or amides of kikumycin derivatives and related synthetic compounds was represented by the elimination of ammonia or water from the molecular ions to give stable nitrile ions. Additionally, the participation of the iminol structure in the pyrrole-2-carboxamide portion of kikumycin A and B was confirmed by the fragment ions derived from their iminol tautomers.     

Mass spectrometric study of camptothecin and related compounds

The electron impact mass spectrometric behavior of ten camptothecin analogs (1-10) is described and discussed in detail with the aid of accurate mass measurement and metastable experiment.     

Preparation of ionic liquid based solid-phase microextraction fiber and its application to forensic determination of methamphetamine and amphetamine in human urine

A new solid-phase microextraction (SPME) procedure using an ionic liquid (IL) has been developed. Reusable IL-based SPME fiber was prepared for the first time by fixing IL through cross-linkage of IL impregnated silicone elastomer on the surface of a fused silica fiber. 1-Ethoxyethyl-3-methylimidazloium bis(trifluoromethane) sulfonylimide ([EeMim][NTf₂]) ionic liquid was employed as a demonstration and the prepared fiber was applied to the forensic headspace determination of methamphetamine (MAP) and amphetamine (AP) in human urine samples. Important extraction parameters including the concentration of salt and base in sample matrix, extraction temperature and extraction time were investigated and optimized. Combined with gas chromatography/mass spectrometry (GC/MS) working in selected ion monitoring (SIM) mode, the new method showed good linearity in the range of 20–1500 μg L⁻¹, good repeatability (RSD < 7.5% for MAP, and <11.5% for AP, n = 6), and low detection limits (0.1 μg L⁻¹ for MAP and 0.5 μg L⁻¹ for AP). Feasibility of the method was evaluated by analyzing human urine samples. Although IL-based SPME is still at the beginning of its development stage, the results obtained by this work showed that it is a promising simple, fast and sensitive sample preparation method.     

One step and highly sensitive headspace solid-phase microextraction sample preparation approach for the analysis of methamphetamine and amphetamine in human urine

A fiber-stable, repeatable and highly sensitive headspace solid-phase microextraction (HS-SPME) method was developed for the analysis of methamphetamine (MA) and amphetamine (AM) in urine using gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. For sample preparation, the test specimen was placed in a 7 ml vial along with the additives (KOH and NaCl) and the internal standards (d8-MA and d8-AM), a glass insert containing heptafluorobutyric anhydride (HFBA) and heptafluorobutyric chloride (HFBCl) as derivatizing reagents was inserted into the vial, the vial was then sealed tightly. A SPME device with a 100 microm polydimethylsiloxane fiber was inserted into the vial and the fiber was exposed to the headspace in the insert, then the vial was heated and stirred at 100 degrees C and 600 rpm for 20 min for evaporation/adsorption/derivatization. The vaporized analytes (AM and MA) in the vial diffused into the glass insert though the holes on the insert, they absorbed onto the fiber, and then interacted with the vapor of the derivatizing reagent. Some of the analytes in the headspace of the glass insert may react with the vapor of the derivatizing reagent first, and then adsorb onto the fiber. The needle was finally removed and inserted into the injection port to desorb the analytes with the fiber exposed to the liner of the GC-MS system for analysis. By combining HFBCl and HFBA as derivatizing reagents and placing them in an insert, the HS-SPME method achieves high sensitivity for the analysis of AM and MA. Correlation coefficients derived from typical calibration curves in the 1.0-1700 ng ml(-1) range are 0.998 for MA and 0.994 for AM. The limits of detection and the limits of quantitation using a sample size of 1 ml are 0.3 and 1.0 ng ml(-1), respectively, for both MA and AM in urine specimens. Because the water hydrolysis of derivatizing reagent is much faster than the acylation reaction of the primary and secondary amines with the derivatizing reagent, the amphetamines cannot be acylated effectively over heated aqueous solution, and therefore this study provides a new acylation design in moisture surroundings. The proposed process also simplifies the procedure for urine sample preparation, and makes the automation of SPME possible.     

Molecularly imprinted solid-phase extraction for the selective determination of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs in human whole blood by gas chromatography-mass spectrometry

A novel method is described for the extraction of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs, such as 3,4-methylenedioxy-methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine, and 3,4-(methylenedioxyphenyl)-2-butanamine, from human whole blood using molecularly imprinted solid-phase extraction as highly selective sample clean-up technique. Whole blood samples were diluted with 10 mmol/L ammonium acetate (pH 8.6) and applied to a SupelMIP-Amphetamine molecularly imprinted solid-phase extraction cartridge. The cartridge was then washed to eliminate interferences, and the amphetamines of interest were eluted with formic acid/methanol (1:100, v/v). After derivatization with trifluoroacetic anhydride, the analytes were quantified using gas chromatography-mass spectrometry. Recoveries of the seven amphetamines spiked into whole blood were 89.1-102%. The limits of quantification for each compound in 200 μL of whole blood were between 0.25 and 1.0 ng. The maximum intra- and inter-day coefficients of variation were 9.96 and 13.8%, respectively. The results show that methamphetamine, amphetamine, and methylenedioxyphenylalkyl-amine designer drugs can be efficiently extracted from crude biological samples such as whole blood by molecularly imprinted solid-phase extraction with good reproducibility. This extraction method will be useful for the pretreatment of human samples before gas chromatography-mass spectrometry.