- Email: [email protected]
MS
910
Abstract / Clinica Chimica Acta 411 (2010) 896–914
3
Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China 4 Department of Pediatrics and Adolescent Medicine, Princess Margaret Hospital, Hong Kong SAR, China 5 Department of Pediatrics, Prince of Wales Hospital, Hong Kong SAR, China 6 Department of Pediatrics and Adolescent Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China 7 Department of Pediatrics, Kwong Wah Hospital, Hong Kong SAR, China 8 Department of Pediatrics and Adolescent Medicine, Caritas Medical Centre, Hong Kong SAR, China
Introduction Dopa-responsive dystonia (DRD) shows promising outcome by simple and effective treatment, therefore, it must be considered in any patients presented with dystonia, idiopathic cerebral palsy, spastic paraplegia, ataxia, and juvenile Parkinsonism. DRD comprises a group of clinically and genetically heterogeneous brain neurotransmitter synthesis disorders. A simple clinical diagnosis of DRD is insufficient to identify the exact underlying disorder that imposes different management. Biochemical and genotype data in Chinese are largely unknown. Methods Twelve unrelated Chinese DRD patients with clinical diagnosis of DRD were included. Six patients had lumbar puncture. Neurotransmitters and pterins were determined in CSF specimens that have been snap-frozen at the bedside on dry ice. They were collected in the first 1 ml because of the rostrocaudal concentration gradient. CSF was frozen at −70 °C until analysis. CSF was analyzed for homovanillic acid (HVA) and 5hydroxyindolacetic acid (5-HIAA) employing HPLC with amperometric electrochemical detector. The oxidized pterins, 5-methyltetrahydrofolic acid (5-MTHF), 3-O-methyl-dopa (3-OMD), and 5-hydroxytryptophan (5-HTP) were analyzed using HPLC with fluorescence detector. Genetic analysis of the pertinent gene was performed. Results Tyrosine hydroxylase deficiency accounts for the majority of DRD (seven of 12 patients). Its age of onset is much younger than other causes. All had low CSF HVA (range 7–133 nmol/l). But, only one patient showed the typical pattern of tyrosine hydroxylase deficiency with low CSF HVA, normal 5-HIAA, low HVA-to-5-HIAA ratio, normal 5-MTHF and pterins. CSF 5-HIAA, pterins and/or 5MTHF concentrations were found to be low in some patients. Four were autosomal dominant GTP cyclohydrolase 1 deficiency and one aromatic L-amino acid decarboxylase deficiency. We characterized 15 different mutations with 11 novel mutations. Conclusions The study illustrates that clinical diagnosis alone is insufficient to predict the genetic pathogenesis which enables proper treatment and accurate genetic counseling. CSF neurotransmitter analysis and genetic analysis are the most decisive diagnostic tools.
doi:10.1016/j.cca.2010.02.048
Poster Quantification of urine oestrogen metabolites using GCMS A.Naimi, B. Anton, A. Read Biochemistry Department, Pathlab, Melbourne, VIC, Australia Email: [email protected] Introduction Urinary oestrogen metabolites may be useful when assessing hormone replacement therapy. Immunoassays are limited in assessing the ratio of oestrogen metabolites and consequently there is increasing demand for chromatographic based assays. Our objective is to use a Hewlett Packard Gas Chromatography mass spectrometry (GCMS) system to identify and quantitate oestrogen metabolites. Methods Five milliliters of urine was mixed with a deuterated internal standard (17β-Estradiol-16,16,17-d3) CDN isotopes (Quebec, Canada). Enzymatic hydrolysis was then used to cleave the oestrogens from their conjugates. A two stage purification process followed: 1. Solid phase extraction with a C18 column; and 2. Liquid extraction with diethyl ether. The purified sample was derivatized with N-trimethylsilylimidazole in pyridine hydrochloride. Five microliters of the derivatized sample was injected onto a HP-1 30 m column (0.25 mm internal diameter and 0.25 µm film) with a helium flow of 0.9 ml/min. The GC temperature gradient was ramped from 180° to 270 °C at a rate of 4°/min followed by 40°/min from 270° to 320 °C. A 5973 MS detector was used in single ion monitoring mode to quantitate the individual oestrogens. Results Fragmentation patterns were compared with known oestrogens (Steraloids, Rhode Island, USA).The oestrogens demonstrated the following mass to charge ratio (m/z): Oestrone (E1)-342 m/z; Equilin340 m/z ; 2OH-E1-430 m/z; 4OH-E1-430 m/z; 16OH-E1-286 m/z; Oestradiol-416 m/z; Oestriol-504 m/z;17β-Estradiol(d3)-419 m/z. Linearity was at least 2000 nmol/l with an analytical sensitivity of 20 nmol/l. Conclusions This method successfully separates the seven main oestrogen metabolites. Sensitivity has been the primary challenge in this method development, which was overcome by the use of large urine volume followed by sample concentration and a large injection volume. This method now replaces the individual immunoassay methods previously used.
doi:10.1016/j.cca.2010.02.049
Poster Screening of drug of abuse in urine by LC/MS/MS C.Y.Yong, W.T. Chua, H.Y. Moy, Y.J. Yao, C.P. Lui Analytical Toxicology Laboratory, Illicit Drugs and Toxicology Division, Applied Sciences Group, Health Sciences Authority, 11 Outram Road 169078, Singapore Email: [email protected]
Abstract / Clinica Chimica Acta 411 (2010) 896–914
Introduction In the screening of drugs of abuse, immunoassay technique is the standard procedure for most clinical and forensic applications. It is rapid and requires very minimal sample preparation. However, it is not specific and only limited range of immunoassay reagents are commercially available for the large variety of drugs. In the present study, we developed a rapid, selective and semi-quantitative screening method for 46 drugs of abuse and their metabolites in urine. The drugs included opiates, amphetamines, benzodiazepines, zolpidem, zopiclone, methadone, buprenorphine, cocaine and ketamine. Method The method involved a simple liquid/liquid extraction of 1 ml urine and 50 ng of internal standard (containing d3-morphine, d6-monoacetylmorphine, d8-methamphetamine, d4-norketamine, d5-nitrazepam, d3-benzoylecgonine and d3-norbuprenorphine) treated with 0.5 ml of buffer (pH ∼ 12), extract with 5 ml 1-chlorobutane and followed by the analysis using the ultra performance liquid with chromatography–tandem mass spectrometry (UPLC/MS/MS). The separation was performed using Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 µm) column and all drugs were successfully separated within 4 min with a total run time of 10 min. The mass detection was performed using multiple reaction monitoring (MRM) in the positive electrospray ionization (ESI) mode. Results and conclusion This method was validated by examining linearity, precision, accuracy, recovery and matrix effects. The performance of the method in the screening of drugs of abuse in urine as compared to the existing immunoassay technique was also evaluated. doi:10.1016/j.cca.2010.02.050 Poster Discordance in PON1 and PON2 single nucleotide polymorphism Determinations between the fully automated Sequenom MALDI-TOF MS MassEXTEND™ and manual MS–PCR assays
911
L55M and Q192R and PON2 C311S, G148A) using the multiplex MALDI-TOF MS MassEXTEND™ assay. Discordant genotypic results were repeated using both methods in a singleton setup. Discordant and allele call drop-out rates were calculated as a percentage of the total number of SNPs analyzed. Results Of the 504 SNP genotypes determined by MassEXTEND™ assay, 28 (5.56%) were found to be discordant with MS–PCR. The allelecalling rate was 96–100% among the 4 SNPs tested. Repeat analysis using singleton reactions corrected 22 MassEXTEND™ and 3 MS–PCR discordant results, reducing the discordance rate to 0.6% (3/504). Conclusions While the MALDI-TOF MS MassEXTEND™ assay is an excellent tool for high throughput genotyping studies, careful method optimization and validation is required if the likelihood of producing erroneous results is to be minimized.
doi:10.1016/j.cca.2010.02.051 Poster HPLC–MS/MS assay for the determination of imatinib and its metabolite CGP74588 concentrations in plasma I.Westley 1, V. Di Fazio 2, R. Vanbinst 2, P Wallemacq 2 Laboratoire de Biochimie Medicale, Université Catholique de Louvain, Brussels, Belgium 2 Department of Clinical Chemistry, University Hospital St. Luc, Brussels, Belgium Email: [email protected] 1
Introduction
P.C.Chan 1,3, B.Y.L. Wong 3, D.E.C. Cole 1,2,3 1 Department of Pathobiology and Laboratory Medicine, University of Toronto, Ontario, Canada 2 Department of Medicine, University of Toronto, Ontario, Canada 3 Department of Clinical Pathology, Sunnybrook Health Sciences Centre, Ontario, Canada M4N3M5 Email: [email protected]
Imatinib mesylate, a tyrosine kinase inhibitor, is primarily metabolized by CYP3A4 to form the active metabolite CGP74588. Due to the polymorphic nature of CYP3A4 and its potential to be induced or inhibited by other compounds, Imatinib is a good candidate for therapeutic drug monitoring. The aim was to develop and validate a HPLC–MS/MS assay for the determination of Imatinib and CGP74588 plasma concentrations.
Introduction
Methods
The Sequenom homogenous MassEXTEND™ assay on the MatrixAssisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) MassARRAY™ platform is a high-throughput, fully automated genotyping system. It allows multiplex genotyping of specific of single nucleotide polymorphisms. The objective of the study was to compare the genotype results generated by the MassEXTEND™ assay with a previously established manual genotyping technique, mutagenically separated PCR (MS–PCR) [Clin Chem 1999; 45:1285–7].
Imatinib and CGP74588 calibrators were prepared in drug free plasma to span the concentration range 20–5000 ng/mL. The internal standard (D8-Imatinib, 100 µg/l) and 0.2 N NaOH (500 µl) was added to all tubes. Ethyl acetate (5 ml) was added to all tubes, then mixed and centrifuged. The solvent layer removed, dried under nitrogen gas, reconstituted with 50:50 methanol/water and 20 µl injected into the HPLC. Mobile phase consisted of 0.1% formic acid in acetonitrile and 2 mM ammonium acetate buffer containing 0.1% formic acid (pH 3.0) (45:55) and pumped at 300 µl/min (rt = 3 min). Separation was achieved using a Waters Xterra C8 column (50 2.1 mm) (t = 50 °C) and mass spectrometry detection was in positive electrospray ionization mode. Compound mass transitions were: D8-Imatinib; 503 N 394.3, 503 N 225, Imatinib; 494.5 N 394.5, 494.5 N 217.2 and CGP74588; 480.4 N 393.8.
Methods 126 DNA samples previously genotyped using the multiplex MS-PCR were analyzed for the Paraoxonase 1 and 2 SNPs (PON1
Abstract / Clinica Chimica Acta 411 (2010) 896–914
3
Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China 4 Department of Pediatrics and Adolescent Medicine, Princess Margaret Hospital, Hong Kong SAR, China 5 Department of Pediatrics, Prince of Wales Hospital, Hong Kong SAR, China 6 Department of Pediatrics and Adolescent Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China 7 Department of Pediatrics, Kwong Wah Hospital, Hong Kong SAR, China 8 Department of Pediatrics and Adolescent Medicine, Caritas Medical Centre, Hong Kong SAR, China
Introduction Dopa-responsive dystonia (DRD) shows promising outcome by simple and effective treatment, therefore, it must be considered in any patients presented with dystonia, idiopathic cerebral palsy, spastic paraplegia, ataxia, and juvenile Parkinsonism. DRD comprises a group of clinically and genetically heterogeneous brain neurotransmitter synthesis disorders. A simple clinical diagnosis of DRD is insufficient to identify the exact underlying disorder that imposes different management. Biochemical and genotype data in Chinese are largely unknown. Methods Twelve unrelated Chinese DRD patients with clinical diagnosis of DRD were included. Six patients had lumbar puncture. Neurotransmitters and pterins were determined in CSF specimens that have been snap-frozen at the bedside on dry ice. They were collected in the first 1 ml because of the rostrocaudal concentration gradient. CSF was frozen at −70 °C until analysis. CSF was analyzed for homovanillic acid (HVA) and 5hydroxyindolacetic acid (5-HIAA) employing HPLC with amperometric electrochemical detector. The oxidized pterins, 5-methyltetrahydrofolic acid (5-MTHF), 3-O-methyl-dopa (3-OMD), and 5-hydroxytryptophan (5-HTP) were analyzed using HPLC with fluorescence detector. Genetic analysis of the pertinent gene was performed. Results Tyrosine hydroxylase deficiency accounts for the majority of DRD (seven of 12 patients). Its age of onset is much younger than other causes. All had low CSF HVA (range 7–133 nmol/l). But, only one patient showed the typical pattern of tyrosine hydroxylase deficiency with low CSF HVA, normal 5-HIAA, low HVA-to-5-HIAA ratio, normal 5-MTHF and pterins. CSF 5-HIAA, pterins and/or 5MTHF concentrations were found to be low in some patients. Four were autosomal dominant GTP cyclohydrolase 1 deficiency and one aromatic L-amino acid decarboxylase deficiency. We characterized 15 different mutations with 11 novel mutations. Conclusions The study illustrates that clinical diagnosis alone is insufficient to predict the genetic pathogenesis which enables proper treatment and accurate genetic counseling. CSF neurotransmitter analysis and genetic analysis are the most decisive diagnostic tools.
doi:10.1016/j.cca.2010.02.048
Poster Quantification of urine oestrogen metabolites using GCMS A.Naimi, B. Anton, A. Read Biochemistry Department, Pathlab, Melbourne, VIC, Australia Email: [email protected] Introduction Urinary oestrogen metabolites may be useful when assessing hormone replacement therapy. Immunoassays are limited in assessing the ratio of oestrogen metabolites and consequently there is increasing demand for chromatographic based assays. Our objective is to use a Hewlett Packard Gas Chromatography mass spectrometry (GCMS) system to identify and quantitate oestrogen metabolites. Methods Five milliliters of urine was mixed with a deuterated internal standard (17β-Estradiol-16,16,17-d3) CDN isotopes (Quebec, Canada). Enzymatic hydrolysis was then used to cleave the oestrogens from their conjugates. A two stage purification process followed: 1. Solid phase extraction with a C18 column; and 2. Liquid extraction with diethyl ether. The purified sample was derivatized with N-trimethylsilylimidazole in pyridine hydrochloride. Five microliters of the derivatized sample was injected onto a HP-1 30 m column (0.25 mm internal diameter and 0.25 µm film) with a helium flow of 0.9 ml/min. The GC temperature gradient was ramped from 180° to 270 °C at a rate of 4°/min followed by 40°/min from 270° to 320 °C. A 5973 MS detector was used in single ion monitoring mode to quantitate the individual oestrogens. Results Fragmentation patterns were compared with known oestrogens (Steraloids, Rhode Island, USA).The oestrogens demonstrated the following mass to charge ratio (m/z): Oestrone (E1)-342 m/z; Equilin340 m/z ; 2OH-E1-430 m/z; 4OH-E1-430 m/z; 16OH-E1-286 m/z; Oestradiol-416 m/z; Oestriol-504 m/z;17β-Estradiol(d3)-419 m/z. Linearity was at least 2000 nmol/l with an analytical sensitivity of 20 nmol/l. Conclusions This method successfully separates the seven main oestrogen metabolites. Sensitivity has been the primary challenge in this method development, which was overcome by the use of large urine volume followed by sample concentration and a large injection volume. This method now replaces the individual immunoassay methods previously used.
doi:10.1016/j.cca.2010.02.049
Poster Screening of drug of abuse in urine by LC/MS/MS C.Y.Yong, W.T. Chua, H.Y. Moy, Y.J. Yao, C.P. Lui Analytical Toxicology Laboratory, Illicit Drugs and Toxicology Division, Applied Sciences Group, Health Sciences Authority, 11 Outram Road 169078, Singapore Email: [email protected]
Abstract / Clinica Chimica Acta 411 (2010) 896–914
Introduction In the screening of drugs of abuse, immunoassay technique is the standard procedure for most clinical and forensic applications. It is rapid and requires very minimal sample preparation. However, it is not specific and only limited range of immunoassay reagents are commercially available for the large variety of drugs. In the present study, we developed a rapid, selective and semi-quantitative screening method for 46 drugs of abuse and their metabolites in urine. The drugs included opiates, amphetamines, benzodiazepines, zolpidem, zopiclone, methadone, buprenorphine, cocaine and ketamine. Method The method involved a simple liquid/liquid extraction of 1 ml urine and 50 ng of internal standard (containing d3-morphine, d6-monoacetylmorphine, d8-methamphetamine, d4-norketamine, d5-nitrazepam, d3-benzoylecgonine and d3-norbuprenorphine) treated with 0.5 ml of buffer (pH ∼ 12), extract with 5 ml 1-chlorobutane and followed by the analysis using the ultra performance liquid with chromatography–tandem mass spectrometry (UPLC/MS/MS). The separation was performed using Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 µm) column and all drugs were successfully separated within 4 min with a total run time of 10 min. The mass detection was performed using multiple reaction monitoring (MRM) in the positive electrospray ionization (ESI) mode. Results and conclusion This method was validated by examining linearity, precision, accuracy, recovery and matrix effects. The performance of the method in the screening of drugs of abuse in urine as compared to the existing immunoassay technique was also evaluated. doi:10.1016/j.cca.2010.02.050 Poster Discordance in PON1 and PON2 single nucleotide polymorphism Determinations between the fully automated Sequenom MALDI-TOF MS MassEXTEND™ and manual MS–PCR assays
911
L55M and Q192R and PON2 C311S, G148A) using the multiplex MALDI-TOF MS MassEXTEND™ assay. Discordant genotypic results were repeated using both methods in a singleton setup. Discordant and allele call drop-out rates were calculated as a percentage of the total number of SNPs analyzed. Results Of the 504 SNP genotypes determined by MassEXTEND™ assay, 28 (5.56%) were found to be discordant with MS–PCR. The allelecalling rate was 96–100% among the 4 SNPs tested. Repeat analysis using singleton reactions corrected 22 MassEXTEND™ and 3 MS–PCR discordant results, reducing the discordance rate to 0.6% (3/504). Conclusions While the MALDI-TOF MS MassEXTEND™ assay is an excellent tool for high throughput genotyping studies, careful method optimization and validation is required if the likelihood of producing erroneous results is to be minimized.
doi:10.1016/j.cca.2010.02.051 Poster HPLC–MS/MS assay for the determination of imatinib and its metabolite CGP74588 concentrations in plasma I.Westley 1, V. Di Fazio 2, R. Vanbinst 2, P Wallemacq 2 Laboratoire de Biochimie Medicale, Université Catholique de Louvain, Brussels, Belgium 2 Department of Clinical Chemistry, University Hospital St. Luc, Brussels, Belgium Email: [email protected] 1
Introduction
P.C.Chan 1,3, B.Y.L. Wong 3, D.E.C. Cole 1,2,3 1 Department of Pathobiology and Laboratory Medicine, University of Toronto, Ontario, Canada 2 Department of Medicine, University of Toronto, Ontario, Canada 3 Department of Clinical Pathology, Sunnybrook Health Sciences Centre, Ontario, Canada M4N3M5 Email: [email protected]
Imatinib mesylate, a tyrosine kinase inhibitor, is primarily metabolized by CYP3A4 to form the active metabolite CGP74588. Due to the polymorphic nature of CYP3A4 and its potential to be induced or inhibited by other compounds, Imatinib is a good candidate for therapeutic drug monitoring. The aim was to develop and validate a HPLC–MS/MS assay for the determination of Imatinib and CGP74588 plasma concentrations.
Introduction
Methods
The Sequenom homogenous MassEXTEND™ assay on the MatrixAssisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) MassARRAY™ platform is a high-throughput, fully automated genotyping system. It allows multiplex genotyping of specific of single nucleotide polymorphisms. The objective of the study was to compare the genotype results generated by the MassEXTEND™ assay with a previously established manual genotyping technique, mutagenically separated PCR (MS–PCR) [Clin Chem 1999; 45:1285–7].
Imatinib and CGP74588 calibrators were prepared in drug free plasma to span the concentration range 20–5000 ng/mL. The internal standard (D8-Imatinib, 100 µg/l) and 0.2 N NaOH (500 µl) was added to all tubes. Ethyl acetate (5 ml) was added to all tubes, then mixed and centrifuged. The solvent layer removed, dried under nitrogen gas, reconstituted with 50:50 methanol/water and 20 µl injected into the HPLC. Mobile phase consisted of 0.1% formic acid in acetonitrile and 2 mM ammonium acetate buffer containing 0.1% formic acid (pH 3.0) (45:55) and pumped at 300 µl/min (rt = 3 min). Separation was achieved using a Waters Xterra C8 column (50 2.1 mm) (t = 50 °C) and mass spectrometry detection was in positive electrospray ionization mode. Compound mass transitions were: D8-Imatinib; 503 N 394.3, 503 N 225, Imatinib; 494.5 N 394.5, 494.5 N 217.2 and CGP74588; 480.4 N 393.8.
Methods 126 DNA samples previously genotyped using the multiplex MS-PCR were analyzed for the Paraoxonase 1 and 2 SNPs (PON1