7 Aminoflunitrazepam

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Mingren Fuh - One of the best experts on this subject based on the ideXlab platform.

  • quantification of 7 Aminoflunitrazepam in human urine by polymeric monolith based capillary liquid chromatography coupled to tandem mass spectrometry
    Talanta, 2018
    Co-Authors: Hsiangyu Liu, Shuling Lin, Mingren Fuh
    Abstract:

    Using a simple liquid-liquid extraction (LLE) procedure for sample pretreatment, 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples by polymeric monolith-based capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The linearity was found in the range of 0.1-50ngmL-1 with a method detection limit (signal-to-noise ratio of 3) estimated at 0.05ngmL-1. Using the proposed method, good precision and recovery were also found in spiked urine samples at the levels of 0.5, 5.0, and 50ngmL-1 (intra-day/inter-day precision: 0.6-1.8% / 0.1-0.8%; post-spiked/pre-spiked recovery: 95.4-102.9% / 96.3-102.5%). In addition, acceptable relative differences (-24.2 - 0.8%) were observed by analyzing clinical urine samples using this monolith-based capillary LC-MS/MS method compared with the results obtained by the routine GC-MC method. Using the monolithic column, no noticeable deterioration of separation efficiency or carry-over was observed for more than 200 injections of urine samples. The applicability of the developed monolith-based capillary LC-MS/MS method was demonstrated by quantifying 7-aminoFM2 in various clinical urine samples. Based on these experimental results, the proposed LLE-monolith-based capillary LC-MS/MS method shows the potential for routine determination of drug metabolites in human urine for clinical and forensic applications.

  • characterization and evaluation of two dimensional microfluidic chip hplc coupled to tandem mass spectrometry for quantitative analysis of 7 Aminoflunitrazepam in human urine
    Analyst, 2010
    Co-Authors: Hsinyu Bai, Shuling Lin, Shenan Chan, Mingren Fuh
    Abstract:

    Microfluidic chip-based high-performance-liquid-chromatography coupled to mass spectrometry (chip-HPLC-MS) has been widely used in proteomic research due to its enhanced sensitivity. We employed a chip-HPLC-MS system for determining small molecules such as drug metabolites in biological fluids. This chip-HPLC-MS system integrates a microfluidic switch, a 2-dimensional column design including an enrichment column (160 nL) for sample pre-concentration and an analytical column for chromatographic separation, as well as a nanospray emitter on a single polyimide chip. In this study, a relatively large sample volume (500 nL) was injected into the enrichment column for pre-concentration and an additional 4 μL of the initial mobile phase was applied to remove un-retained components from the sample matrix prior to chromatographic separation. The 2-dimensional column design provides the advantages of online sample concentration and reducing matrix influence on MS detection. 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples using the integrated chip-HPLC-MS system. The linear range was 0.1–10 ng mL−1 and the method detection limit (signal-to-noise ratio of 3) was 0.05 ng mL−1 for 7-aminoFM2. After consecutive liquid–liquid extraction (LLE) and solid-phase extraction (SPE), the chip-HPLC-MS exhibited high correlation between 7-aminoFM2 spiked Milli-Q water and 7-aminoFM2 spiked urine samples. This system also showed good precision (n = 5) and recovery for spiked urine samples at the levels of 0.1, 1.0, and 10 ng mL−1. Intra-day and inter-day precision were 2.0–7.1% and 4.3–6.0%, respectively. Clinical urine samples were also analyzed by this chip-HPLC-MS system and acceptable relative differences (−1.3 to −13.0%) compared with the results using a GC-MC method were determined. Due to its high sensitivity and ease of operation, the chip-HPLC-MS system can be utilized for the determination of small molecules such as drug metabolites and neurotransmitters in biological fluids for clinical diagnosis.

  • determination of 7 Aminoflunitrazepam in urine by dispersive liquid liquid microextraction with liquid chromatography electrospray tandem mass spectrometry
    Talanta, 2009
    Co-Authors: Mahaveer B Melwanki, Weishan Chen, Hsinyu Bai, Tzuenyeuan Lin, Mingren Fuh
    Abstract:

    Dispersive liquid-liquid microextraction (DLLME) and liquid chromatography-electrospray-tandem mass spectrometry (LC-ES-MS/MS) procedure was presented for the extraction and determination of 7-Aminoflunitrazepam (7-aminoFM2), a biomarker of the hypnotic flunitrazepam (FM2) in urine sample. The method was based on the formation of tiny droplets of an organic extractant in the sample solution using water-immiscible organic solvent [dichloromethane (DCM), an extractant] dissolved in water-miscible organic dispersive solvent [isopropyl alcohol (IPA)]. First, 7-aminoFM2 from basified urine sample was extracted into the dispersed DCM droplets. The extracting organic phase was separated by centrifuging and the sedimented phase was transferred into a 300 microl vial insert and evaporated to dryness. The residue was reconstituted in 30 microl mobile phase (20:80, acetonitrile:water). An aliquot of 20 microl as injected into LC-ES-MS/MS. Various parameters affecting the extraction efficiency (type and volume of extraction and dispersive solvent, effect of alkali and salt) were evaluated. Under optimum conditions, precision, linearity (correlation coefficient, r(2)=0.988 over the concentration range of 0.05-2.5 ng/ml), detection limit (0.025 ng/ml) and enrichment factor (20) had been obtained. To our knowledge, DLLME was applied to urine sample for the first time.

Jan Tytgat - One of the best experts on this subject based on the ideXlab platform.

  • development and validation of a fast ionic liquid based dispersive liquid liquid microextraction procedure combined with lc ms ms analysis for the quantification of benzodiazepines and benzodiazepine like hypnotics in whole blood
    Forensic Science International, 2017
    Co-Authors: Marieke De Boeck, Jan Tytgat, Sophie Missotten, Wim Dehaen, Eva Cuypers
    Abstract:

    To date, thorough clean-up of complex biological samples remains an essential part of the analytical process. The solid phase extraction (SPE) technique is the well-known standard, however, its main weaknesses are the labor-intensive and time-consuming protocols. In this respect, dispersive liquid-liquid microextractions (DLLME) seem to offer less complex and more efficient extraction procedures. Furthermore, ionic liquids (ILs) - liquid salts - are emerging as new promising extraction solvents, thanks to their non-flammable nature, negligible vapor pressure and easily adaptable physiochemical properties. In this study, we investigated whether ILs can be used as an extraction solvent in a DLLME procedure for the extraction of a broad range of benzodiazepines and benzodiazepine-like hypnotics in whole blood samples. 1.0mL whole blood was extracted using an optimized 30-min IL-based DLLME procedure, followed by LC-ESI(+)-MS/MS analysis in scheduled MRM scan mode. The optimized analytical method was successfully validated for 7-Aminoflunitrazepam, alprazolam, bromazepam, clobazam, clonazepam, clotiazepam, diazepam, estazolam, ethyl loflazepate, etizolam, flurazepam, lormetazepam, midazolam, oxazepam, prazepam, temazepam, triazolam, zolpidem and zopiclone. The method showed good selectivity for endogenous interferences based on 12 sources of blank whole blood. No benzodiazepine interferences were observed, except for clorazepate and nordiazepam, which were excluded from the quantitative method. Matrix-matched calibration curves were constructed covering the whole therapeutic range, including low toxic plasma concentrations. Accuracy and precision results met the proposed acceptance criteria for the vast majority of compounds, except for brotizolam, chlordiazepoxide, cloxazolam, flunitrazepam, loprazolam, lorazepam and nitrazepam, which can only be determined in a semi-quantitative way. Recoveries were within the range of 24.7%-127.2% and matrix effects were within 20.0%-92.6%. Both parameters were tested using 5 sources of whole blood and coefficients of variance were below 20%. Overall, the applicability of ILs as promising solvents for the extraction of benzodiazepines in whole blood samples has been proven. Moreover, a fast and easy IL-based DLLME procedure was developed for the quantification of 19 benzodiazepines and benzodiazepine-like hypnotics.

  • The evaluation of the applicability of a high pH mobile phase in ultrahigh performance liquid chromatography tandem mass spectrometry analysis of benzodiazepines and benzodiazepine-like hypnotics in urine and blood
    Journal of Chromatography A, 2012
    Co-Authors: Ruth Verplaetse, Eva Cuypers, Jan Tytgat
    Abstract:

    Abstract A sensitive liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous detection of benzodiazepines, benzodiazepine-like hypnotics and some metabolites (7-Aminoflunitrazepam, alprazolam, bromazepam, brotizolam, chlordiazepoxide, chlornordiazepam, clobazam, clonazepam, clotiazepam, cloxazolam, diazepam, ethylloflazepate, flunitrazepam, flurazepam, loprazolam, lorazepam, lormetazepam, midazolam, N -desmethylflunitrazepam, nitrazepam, N -methylclonazepam (internal standard), nordiazepam, oxazepam, prazepam, temazepam, tetrazepam, triazolam, zaleplon, zolpidem, zopiclone) in urine and whole blood. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction cartridge. Electrospray ionization was found to be more efficient than atmospheric pressure chemical ionization. The use of a mobile phase of high pH resulted in higher retention and higher electrospray ionization signals than the conventional low pH mobile phases. Considering the benefits of a high pH mobile phase on both chromatography and mass spectrometry, its use should be encouraged. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed on a small particle column (Acquity C18, 1.7 μm, 2.1 mm × 50 mm). The optimized method was fully validated.

Eva Cuypers - One of the best experts on this subject based on the ideXlab platform.

  • development and validation of a fast ionic liquid based dispersive liquid liquid microextraction procedure combined with lc ms ms analysis for the quantification of benzodiazepines and benzodiazepine like hypnotics in whole blood
    Forensic Science International, 2017
    Co-Authors: Marieke De Boeck, Jan Tytgat, Sophie Missotten, Wim Dehaen, Eva Cuypers
    Abstract:

    To date, thorough clean-up of complex biological samples remains an essential part of the analytical process. The solid phase extraction (SPE) technique is the well-known standard, however, its main weaknesses are the labor-intensive and time-consuming protocols. In this respect, dispersive liquid-liquid microextractions (DLLME) seem to offer less complex and more efficient extraction procedures. Furthermore, ionic liquids (ILs) - liquid salts - are emerging as new promising extraction solvents, thanks to their non-flammable nature, negligible vapor pressure and easily adaptable physiochemical properties. In this study, we investigated whether ILs can be used as an extraction solvent in a DLLME procedure for the extraction of a broad range of benzodiazepines and benzodiazepine-like hypnotics in whole blood samples. 1.0mL whole blood was extracted using an optimized 30-min IL-based DLLME procedure, followed by LC-ESI(+)-MS/MS analysis in scheduled MRM scan mode. The optimized analytical method was successfully validated for 7-Aminoflunitrazepam, alprazolam, bromazepam, clobazam, clonazepam, clotiazepam, diazepam, estazolam, ethyl loflazepate, etizolam, flurazepam, lormetazepam, midazolam, oxazepam, prazepam, temazepam, triazolam, zolpidem and zopiclone. The method showed good selectivity for endogenous interferences based on 12 sources of blank whole blood. No benzodiazepine interferences were observed, except for clorazepate and nordiazepam, which were excluded from the quantitative method. Matrix-matched calibration curves were constructed covering the whole therapeutic range, including low toxic plasma concentrations. Accuracy and precision results met the proposed acceptance criteria for the vast majority of compounds, except for brotizolam, chlordiazepoxide, cloxazolam, flunitrazepam, loprazolam, lorazepam and nitrazepam, which can only be determined in a semi-quantitative way. Recoveries were within the range of 24.7%-127.2% and matrix effects were within 20.0%-92.6%. Both parameters were tested using 5 sources of whole blood and coefficients of variance were below 20%. Overall, the applicability of ILs as promising solvents for the extraction of benzodiazepines in whole blood samples has been proven. Moreover, a fast and easy IL-based DLLME procedure was developed for the quantification of 19 benzodiazepines and benzodiazepine-like hypnotics.

  • The evaluation of the applicability of a high pH mobile phase in ultrahigh performance liquid chromatography tandem mass spectrometry analysis of benzodiazepines and benzodiazepine-like hypnotics in urine and blood
    Journal of Chromatography A, 2012
    Co-Authors: Ruth Verplaetse, Eva Cuypers, Jan Tytgat
    Abstract:

    Abstract A sensitive liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous detection of benzodiazepines, benzodiazepine-like hypnotics and some metabolites (7-Aminoflunitrazepam, alprazolam, bromazepam, brotizolam, chlordiazepoxide, chlornordiazepam, clobazam, clonazepam, clotiazepam, cloxazolam, diazepam, ethylloflazepate, flunitrazepam, flurazepam, loprazolam, lorazepam, lormetazepam, midazolam, N -desmethylflunitrazepam, nitrazepam, N -methylclonazepam (internal standard), nordiazepam, oxazepam, prazepam, temazepam, tetrazepam, triazolam, zaleplon, zolpidem, zopiclone) in urine and whole blood. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction cartridge. Electrospray ionization was found to be more efficient than atmospheric pressure chemical ionization. The use of a mobile phase of high pH resulted in higher retention and higher electrospray ionization signals than the conventional low pH mobile phases. Considering the benefits of a high pH mobile phase on both chromatography and mass spectrometry, its use should be encouraged. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed on a small particle column (Acquity C18, 1.7 μm, 2.1 mm × 50 mm). The optimized method was fully validated.

Pascal Kintz - One of the best experts on this subject based on the ideXlab platform.

  • segmental hair analysis can demonstrate external contamination in postmortem cases
    Forensic Science International, 2012
    Co-Authors: Pascal Kintz
    Abstract:

    Abstract Excluding laboratory mistakes, a false positive hair result can be observed in case of contamination from environmental pollution (external contamination) or after drug incorporation into the hair from the individual body fluids, such as sweat or putrefactive fluid (post mortem artifact). From our 20 years experience of hair testing, it appears that artifact(s) cannot be excluded in some post mortem cases, despite a decontamination procedure. As a consequence, interpretation of the results is a challenge that deserves particular attention. Our strategy will be reviewed in this paper, based on six cases. In all cases, a decontamination procedure with two washes of 5 ml of dichloromethane for 5 min was performed and the last dichloromethane wash was negative for each target drug. From the histories, there was no suspicion of chronic drug use. In all six cases, the concentrations detected were similar along the hair shaft, irrespective of the tested segment. We have considered this as indicative of external contamination and suggested to the forces or the judges that it is not possible to indicate exposure before death. In contrast to smoke, it seems that contamination due to aqueous matrices (sweat, putrefactive fluid, blood) is much more difficult to remove. To explain potential incorporation of 7-Aminoflunitrazepam via putrefactive material, the author incubated negative hair strands in blood spiked at 100 ng/ml and stored at +4 °C, room temperature and +40 °C for 7, 14 and 28 days. After routine decontamination, 7-Aminoflunitrazepam tested positive in hair, irrespective of the incubation temperature, as early as after 7 days (233–401 pg/mg). In all periods, maximum concentrations were observed after incubation at room temperature. The highest concentration (742 pg/mg) was observed after 28 days incubation at room temperature. It is concluded that a standard decontamination procedure is not able to completely remove external contamination in case of post mortem specimens. Homogenous segmental analyses can be probably indicative of external contamination and therefore a single hair result should not be used to discriminate long-term exposure to a drug. Nor should the presence of a metabolite be considered as a discrimination tool, as it can also be present in putrefactive material.

  • External post mortem artefact: a key issue in hair result interpretation
    'EDP Sciences', 2009
    Co-Authors: Pascal Kintz, Marion Villain, Vincent Cirimele
    Abstract:

    Purpose: Excluding laboratory mistakes, a false positive hair result can be observed in case of contamination from environmental pollution (external contamination) or after drug incorporation into the hair from the individual body fluids, such as sweat or putrefactive fluid (post mortem artefact). From our 18 years experience of hair testing, it appears that artefact(s) cannot be excluded in some post mortem cases, despite a decontamination procedure. As a consequence, interpretation of the results is a challenge that deserves particular attention. Our strategy will be reviewed in this paper. Methods: Three authentic cases are presented to document our hypothesis. Results: Case 1: a 24-year old man was found dead in a friend's house. He was not known as a drug addict. The analysis of femoral blood was interpreted as ecstasy poisoning (MDMA = 770 ng/mL, MDA = 56 ng/mL). Segmental hair analysis (GC/MS) was as follows: MDMA = 0.94, 0.87 and 0.90 ng/mg in the 0–3, 3–6 and 6–9 cm, respectively. No MDA was detected. Case 2: at the time of death, cyamemazine, which was never prescribed to the subject, was detected in femoral blood at 3660 ng/mL. The body was exhumed 18 months after burial. Segmental hair cyamemazine analysis (LC/MS-MS) was as follows: 3.1 ng/mg (0–2 cm), 2.9 ng/mg (2–4 cm) and 3.1 ng/mg (4–6 cm). Case 3: the skeleton of a young girl was found in a water well 20 years after her disappearance. 7-amino-flunitrazepam was detected in her cerebral material at 0.67 ng/g. Some hair fibers, attached to the skull were collected. Segmental hair 7-Aminoflunitrazepam analysis (LC/MS-MS) was as follows: 15 pg/mg (0–2 cm) and 19 pg/mg (2–4 cm). In all cases, a decontamination procedure with 2 washes of 5 mL of dichloromethane for 5 min was achieved and the last dichloromethane bath was negative for each target drug. From the histories, there was no suspicion of chronic drug use. In all cases, the concentrations detected were homogenous, irrespective of the tested segment. This can be considered as good indicative of potential external contamination. In contrast to smoke, it seems that contamination due to aqueous matrices (sweat, putrefactive fluid, blood) is much more difficult to remove. To explain potential incorporation of 7-Aminoflunitrazepam via putrefactive material, the authors incubated negative hair strands in blood spiked at 100 ng/mL and stored at +4 °C, room temperature and +40 °C for 7, 14 and 28 days. After routine decontamination, 7-Aminoflunitrazepam tested positive in hair, irrespective of the incubation temperature, as early as after 7 days (233–401 pg/mg). In all periods, maximum concentrations were observed after incubation at room temperature. The highest concentration (742 pg/mg) was observed after 28 days incubation at room temperature. Conclusion: It is concluded that a standard decontamination procedure is not able to completely remove external contamination in case of post mortem specimens. Homogenous segmental analyses can be probably indicative of external contamination and therefore a single hair result should not be used to discriminate long-term exposure to a drug. The presence of a metabolite should not be considered as a discrimination tool, as it can also be present in putrefactive material

  • detection du 7 Aminoflunitrazepam dans de la matiere cerebrale par uplc tof ms
    Annales De Toxicologie Analytique, 2008
    Co-Authors: Pascal Kintz, Guillaume Salquebre, Marion Villain, Vincent Cirimele
    Abstract:

    Dans le cadre d’une expertise toxicologique, la recherche de molecules sedatives, hypnotiques ou tranquillisantes dans de la matiere cerebrale en decomposition a ete requise. L’echantillon provenait d’une tete humaine retrouvee dans un sac plastique et qui aurait sejourne au fond d’un puits pendant pres d’une quinzaine d’annees. L’investigation a ete menee par chromatographie liquide couplee a la spectrometrie de masse haute resolution avec acquisition de spectres complets. Un logiciel permet la detection et le dosage semi-quantitatif de composes dans des matrices complexes a l’aide d’une librairie etablie a partir des composes standards apres injection. L’identification est basee sur les criteres de temps de retention, de masse exacte, de rapports isotopiques et de presence d’ions de fragments caracteristiques. Le couplage chromatographie liquide et temps de vol est une technologie emergente, qui permet en theorie le criblage large des xenobiotiques, a partir d’une librairie maison, basee sur l’enregistrement de spectres moleculaires avec une resolution de l’ordre de 10 000. Quelques rares applications sont disponibles a ce jour dans la litterature, essentiellement pour les urines [1–5].

  • screening method for benzodiazepines and hypnotics in hair at pg mg level by liquid chromatography mass spectrometry mass spectrometry
    Journal of Chromatography B, 2005
    Co-Authors: Marion Villain, Vincent Cirimele, Marta Concheiro, Pascal Kintz
    Abstract:

    Abstract A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LC–MS/MS (alprazolam, 7-aminoclonazepam, 7-Aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpidem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid–liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography–tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20-mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r2 > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.

  • screening and confirmatory method for benzodiazepines and hypnotics in oral fluid by lc ms ms
    Forensic Science International, 2005
    Co-Authors: Pascal Kintz, Marion Villain, Marta Concheiro, Vincent Cirimele
    Abstract:

    A procedure is presented for the screening of 17 benzodiazepines and hypnotics in oral fluid after collection with the Intercept(R) device by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-Aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon, zopiclone and zolpidem). The method involves extraction of 0.5 mL of oral fluid (previously stored in the Intercept blue buffer) treated with 0.5 mL of phosphate buffer (pH 8.4) in the presence of 5 ng diazepam-d(5) used as internal standard, with 3 mL of diethyl ether/methylene chloride (50/50) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.1 to 0.2 ng/mL. Linearity is observed from the limit of quantification of each compound to 20 ng/mL (r(2)>0.99). Coefficients of variation at 2 ng/mL, measured on 6 points range from 4 to 8% for all drugs, except zopiclone (34%). Extraction recovery, measured at the same concentration was higher than 90%. Ion suppression was evaluated for each compound and was lower than 10% for all drugs except zopiclone (93%). These results were found suitable to screen for 17 benzodiazepines in oral fluid and detect them at very low concentrations, making this method suitable for monitoring subjects under the influence.

Hsinyu Bai - One of the best experts on this subject based on the ideXlab platform.

  • characterization and evaluation of two dimensional microfluidic chip hplc coupled to tandem mass spectrometry for quantitative analysis of 7 Aminoflunitrazepam in human urine
    Analyst, 2010
    Co-Authors: Hsinyu Bai, Shuling Lin, Shenan Chan, Mingren Fuh
    Abstract:

    Microfluidic chip-based high-performance-liquid-chromatography coupled to mass spectrometry (chip-HPLC-MS) has been widely used in proteomic research due to its enhanced sensitivity. We employed a chip-HPLC-MS system for determining small molecules such as drug metabolites in biological fluids. This chip-HPLC-MS system integrates a microfluidic switch, a 2-dimensional column design including an enrichment column (160 nL) for sample pre-concentration and an analytical column for chromatographic separation, as well as a nanospray emitter on a single polyimide chip. In this study, a relatively large sample volume (500 nL) was injected into the enrichment column for pre-concentration and an additional 4 μL of the initial mobile phase was applied to remove un-retained components from the sample matrix prior to chromatographic separation. The 2-dimensional column design provides the advantages of online sample concentration and reducing matrix influence on MS detection. 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples using the integrated chip-HPLC-MS system. The linear range was 0.1–10 ng mL−1 and the method detection limit (signal-to-noise ratio of 3) was 0.05 ng mL−1 for 7-aminoFM2. After consecutive liquid–liquid extraction (LLE) and solid-phase extraction (SPE), the chip-HPLC-MS exhibited high correlation between 7-aminoFM2 spiked Milli-Q water and 7-aminoFM2 spiked urine samples. This system also showed good precision (n = 5) and recovery for spiked urine samples at the levels of 0.1, 1.0, and 10 ng mL−1. Intra-day and inter-day precision were 2.0–7.1% and 4.3–6.0%, respectively. Clinical urine samples were also analyzed by this chip-HPLC-MS system and acceptable relative differences (−1.3 to −13.0%) compared with the results using a GC-MC method were determined. Due to its high sensitivity and ease of operation, the chip-HPLC-MS system can be utilized for the determination of small molecules such as drug metabolites and neurotransmitters in biological fluids for clinical diagnosis.

  • determination of 7 Aminoflunitrazepam in urine by dispersive liquid liquid microextraction with liquid chromatography electrospray tandem mass spectrometry
    Talanta, 2009
    Co-Authors: Mahaveer B Melwanki, Weishan Chen, Hsinyu Bai, Tzuenyeuan Lin, Mingren Fuh
    Abstract:

    Dispersive liquid-liquid microextraction (DLLME) and liquid chromatography-electrospray-tandem mass spectrometry (LC-ES-MS/MS) procedure was presented for the extraction and determination of 7-Aminoflunitrazepam (7-aminoFM2), a biomarker of the hypnotic flunitrazepam (FM2) in urine sample. The method was based on the formation of tiny droplets of an organic extractant in the sample solution using water-immiscible organic solvent [dichloromethane (DCM), an extractant] dissolved in water-miscible organic dispersive solvent [isopropyl alcohol (IPA)]. First, 7-aminoFM2 from basified urine sample was extracted into the dispersed DCM droplets. The extracting organic phase was separated by centrifuging and the sedimented phase was transferred into a 300 microl vial insert and evaporated to dryness. The residue was reconstituted in 30 microl mobile phase (20:80, acetonitrile:water). An aliquot of 20 microl as injected into LC-ES-MS/MS. Various parameters affecting the extraction efficiency (type and volume of extraction and dispersive solvent, effect of alkali and salt) were evaluated. Under optimum conditions, precision, linearity (correlation coefficient, r(2)=0.988 over the concentration range of 0.05-2.5 ng/ml), detection limit (0.025 ng/ml) and enrichment factor (20) had been obtained. To our knowledge, DLLME was applied to urine sample for the first time.