Determination of Barbiturates in urine by GC/MS

Xu Qin

（Department of Clinical Laboratory Science,Zunyi Meidcal College,Guizhou Zunyi 563003, China）

Determination of Barbiturates in urine by GC/MS

Xu Qin

（Department of Clinical Laboratory Science,Zunyi Meidcal College,Guizhou Zunyi 563003, China）

ObjectiveTo introduce a highly selective and sensitive procedure for isolating and identifying barbiturates in human urin established by University of kentucky,USA.Methods①An aliquot of urine is extracted with a Toxi-tube and derivatized with N,N-dimethylformamide dimethyl acetal（DMF-DMA）.②The sample is analyzed by GC/MS and selected ion monitoring mode （SIM）was used for identification Drug Quant software was used for quantification.for the following barbiturates:butalbital,amobarbital,pentobarbital,secobarbital and phenobarbital in human urin.Results①The cutoff level for GC/MS is 100 ng/ml for amobarbital,butalbital,pentobarbital,phenobarbital and 30 ng/mL for secobarbital.②The method/procedure also showed high between run and within run precision and accuracy.ConlusionsThis method /procedure is more selective，more sensitive，simpler，more accurate，and can be applied to clinical diagnosis of barbiturates poisoning and barbiturates abuse.

Barbiturates;GC/MS；urine sample

Barbiturates produce a wide spectrum of central nervous systerm depression from mild sedation to coma and have been used as sedatives,hypnotics,anesthetics and anticonvelsants.Barbiturates are classified as ultrashort,short,intermediate and long-acting.Barbiturates are common drug of abuse taken orally or intravenously.Chronic use will develip tolerance,physical dependence and psychological dependence on barbiturates. Overdoses can cause profound shock,coma,or death. Shorting acting barbiturates can be detected for only 1～4 days,while long-acting barbiturates（Bartital,phenbarbital）can be detected for 2～3 weeks.

Some labratories utilize immunoassay （EIA）for detecting barbiturates in urine.The immunoassay is quick,highly sensitive,and relatively inexpensive but may lack specificity.Positive confirmation tests may occur in urine specimens from patients who legally or unknowingly ingest products that contain drugs of abuse.In these instances,the finding is a true positive but may not reflect drug abuse by the client.Gas chromatography in combination with mass spectrometry （GC-MS）is a more expensive and time-consuming test,but is the gold standard for confirming a positive result on immunoassay.

1 Instrumentation and Reagent

1.1 Instrumentation An Agilent 6890N gas chromatograph with a fused silica capillary column [25 m x 0.2 mm i.d.,0.33 μm film thickness:cross-linked 5% phenylmethyl silicon （Agilent P/N 19091-B option 102）]coupled to a mass selective detector,model 5975；Quantitation is achieved with the Drug Quant誖software on the Agilent G1039C MS Chemstation；Reacti-therm Heating module （Pierce Chemical Co.） with associated Reacti-block.

1.2 Reagent

1.2.1 Stock Reagents N,N-Dimethylformamide（DMF）, （Fisher Scientific Certified ACS D119-1）;N,NDimethylformamide dimethyl acetal（DMF-DMA）,（ Sigma-Aldrich ChemicalCo.,#14,073-2）;Hexane, HPLC Grade（Fisher Scientific）;Methanol（HPLC Grade （Fisher Scientific）;Sodium Acetate Trihydrate,（Sigma-Aldrich Chemical Co,#S-8625）;HCl Concentrate, ACS grade（Fisher Scientific）;Pentobarbital（1.0 mg/mL, Cerilliant,P-010）;Secobarbital（1.0 mg/mL,Cerilliant, S-002）;Butalbital（1.0 mg/ml,Cerilliant,B-006）;Am-obarbital（1.0 mg/mL,Cerilliant,A-020）;Phenobarbital （1.0 mg/mL.Cerilliant,P-008）;Phenobarbital-d5（Cerilliant,P-018）;Pentobarbital.-d5（Cerilliant,P-009）

1.2.2 Working Reagents

HCl 0.2 mol/L;Acetate Buffer,pH 7.3.

2 Control and Standards

2.1 Control BioRad Liquichek urine toxicology control 2.2 Standards

2.2.1 Barbiturate combined intermediate standard,10 μg/mL；100 ng/mL；500 ng/mL；1000 ng/mL；Internal standard phenobarbital-d5and pentobarbital-d5,25 μg/ mL

3 Methods and Procedure

3.1 Sample Requirements:A 4 mL aliquot of randomly collected urine.

3.2 Complete Column and Detector Performance section for the indicated standard.

3.3 Sample extraction derivatization and analysis:

3.3.1 Add 4.0 mL of working standard,control and unknown to properly labeled Toxi-tube B extraction tubes.

3.3.2 Add 100 μL internal standard to each and mix by inversion for one minute.

3.3.3 Centrifuge for five minutes and transfer the organic phase to labeled 5 mL disposable centrifuge tubes.

3.3.4 Evaporate organic solvent under a stream of air at 37℃.

3.3.5 To the dried extract add 100 μL DMF and 100 μL DMF-DMA.

3.3.6 Incubate the tube at 130℃for 2～5 minutes.

3.3.7 Cool the tubes under a stream of cold water for 15 seconds.Add 1.0 mL acetate buffer.

3.3.8 Add 1 mL hexane,cap and vortex well.

3.3.9 Transfer 100 μL hexane to autosampler vial with insert and cap vial.

3.3.10 Refer to Agilent MSD Chemstation D.02.00.275 Software Guide for sample analysis details.

4 Results

4.1 Sample Identification The information listed below is an aid to identify and quantitate analytes of interest （table 1）.

Tab 1 Sample Identification

4.2 Criteria For The Acceptance of Results Acceptance of the standard curve is as follows:R2≥ 0.998; QC within established limits.

4.3 Results Reporting

4.3.1 The cut-off values for reporting barbiturates are as follows：Amobarbital 100 ng/mL；Butalbita100 ng/ mL；Butabarbital100 ng/mL；Pentobarbital100 ng/mL；Phenobarbital 100 ng/mL；Secobarbital 30 ng/mL.Results below these cut-off's are reported as negative.

4.3.2 The upper limits of linearity for the barbiturates are as follows:Amobarbital 2000 ng/mL;Butalbital 2000 ng/mL;Pentobarbital 2000 ng/mL;Phenobarbital 2000 ng/mL;Secobarbital 2000 ng/mL.Results above the upper limit of linearity are reported as>2000 ng/ mL.

5 Discussions and Clinical Significance

The barbiturates have a low therapeutic index and a relatively high abuse potential.Because of their rapid onset and short duration of action,the short to intermediate acting barbiturates are used as sedativehypnotics（amobarbital,butabarbital,butalbital,pentobarbital,secobarbital）and are those most commonly abused.The longer acting barbiturates（mephobarbital, phenobarbital）,used primarily for their anticonvulsant properties,are rarely abused.

The barbiturates undergo extensive hepatic metabolism in which the C5 substituents are transformed to alcohols,phenols,ketones,or carboxylic acids;these metabolites may be excreted in urine in part as glucuronide conjugates.For some barbiturates（amobarbital, phenobarbital）,N-glucosylation is an additional important metabolic transformation.As a result only a relatively small amount of an administered barbiturate dose is excreted in urine as parent drug;notable exceptions are phenobarbital and aprobarbital.Nevertheless,the parent drugs,rather than hydroxy or carboxylic acid metabolites,are targeted for detection in urine screening and confirmation procedures.This analytical approach is generally successful for barbiturates because these drugs are ingested in sufficiently high doses to allow detection of unmetabolized drug in urine.

[1] Porter WH.Clinical Toxicology.In Tietz Textbook of Clinical Chemistry [M].3rd ed..CA Burtis and ER Ashwood,Eds Philadelphia,W.B.Saunders Co.,1999.

[2] Barbour AD.GC/MS analysis of propylated barbiturates[J]. J Anal Toxicol,1991,15:214-215.

李 勇]

GC/MS法測定尿中的巴比妥酸鹽

許琴

（遵義醫學院臨床檢驗系，貴州遵義563003）

目的 建立高特異性﹑高敏感性可分離及鑒別人體尿液中巴比妥類藥物的程序方法。方法 ①用Toxitube提取人體尿液中的N,N-二甲基甲酰胺二甲基縮醛衍生物（DMF-DMA）。②運用GC/MS﹑SIM系統及藥品定量軟件系對人體尿液中巴比妥類藥物：布他比妥，異戊巴比妥，戊巴比妥，司巴比妥，苯巴比妥進行定性定量分析。結果①GC/MS對人體尿液中司巴比妥最低檢出濃度為30 ng/mL，對布他比妥﹑異戊巴比妥﹑戊巴比妥﹑苯巴比妥最低檢出濃度為200 ng/ml。②在實驗的運行操作中該方法/程序顯示很高的精確度和準確度。結論 此檢測方法選擇性好﹑檢測簡便﹑靈敏﹑定性定量結果準確，可用于巴比妥類藥物中毒及巴比妥類藥物濫用的臨床檢驗診斷。

巴比妥類；氣相色譜/質譜；尿液樣品

R446.12

A

1000-2715（2010）01-0001-03