| Objective To establish the method of auto headspace gas chromatography-mass spectrometry (HS-GC-MS) for the determination of alcohol in blood samples. To study the stability of venous blood and preservative free cardiac blood samples in different storage conditions, investigate the change of the concentration of alcohol in blood samples in different conditions. And analyze pollution of the disinfection process of venous blood in blood samples, to explore the impact of skin disinfectant venipuncture procedures on blood alcohol concentration detection. To identify the process of determination of ethanol in human blood research, evaluate the sources of measurement uncertainty for the by HS-GC-MS. It also established a HS-GC-MS determination of blood alcohol content of quality control chart, to carry out the quality control to provide protection for the laboratory routine drunk driving test results.Methods (1) Alcohol determination in human blood by HS-GC-MS: ①Sample preparation:two hundred microliters of blood sample and eight hundred microliters of tert-butanol (Internal standard) standard solution were placed in a 20mL headspace vial, which was immediately sealed with a silicone-septum cap. ② Automatic headspace conditions:Vapor-liquid equilibrium temperature:60℃; duration for vapor-liquid equilibrium:20min. The transfer line temperature and the injection transfer line temperature were 100℃ and 90℃ respectively. The sampling interval was 3min, The times of headspace vial pressing, sample loading and sample injection were 10 s. ③Gas chromatographic conditions were as follow: Chromatograph column:capillary column Rxi-5MS,30m×0.25mm × 0.25μm, carrier gas:high-purified helium (Purity> 99.999%),1.0ml/min(constant flow); inlet:150 ℃, injection mode:split; column temperature:70℃ hold for 1.8 min. ④MS conditions:ion source temperature and transfer line temperature were all 250℃; ionization method was electron ionization with the ionization energy of 70eV, the solvent cut removal time was 1.2min; SIM selected ions monitoring:ethanol (m/z:29,31,45,46) and tert-butyl alcohol (m/z:41,43,57,59) with the scanning interval of 0.1s.(2) Stability analysis of blood samples: ① Subjects:selected drunk driving samples,20 cases of heart blood samples which were placed in ordinary plastic pipe and two cases of venous blood which were sealed in formal blood collection tube. Established methods were used to detect the initial blood alcohol content. ￢enous blood samples experimental procedure:venous blood were stored at room temperature (25℃), and the blood alcohol concentrations were measured at 0,24, 48 and 72 hours.③Heart blood samples experimental procedure:20 samples were taken in three tubes sealed in EP tubes, each case 750uL. Group 1 stored at-18℃ in a freezing container for preservation; group 2 stored at 4℃ in a refrigerator; group 3 storage at room temperature about 25 ℃. The blood alcohol concentrations were measured at 2,6,12,24,168 (7 days),336 (14 days) and 720 hours (30 days). Standard curve were accompanied by each measurement, standard quality control sample was added after each ten samples. SPSS20.0 statistical analysis was used to handles the results.(3) Analyzed the pollution sources of blood ethanol concentration:The blood contamination of ethanol may come from the disinfectant. Thus in the present study, Type Ⅱ Anerdian (containing 65 to 75% ethanol) was used as a skin disinfectant before sampling the venous blood, and chlorhexidine acetate (ethanol-free) was used as control. ①Subjects and conditions:30 volunteers were randomly divided into three groups, and each group consisted of five men and women, with the ages ranged from 20 to 55 years old, healthy and no liver and kidney disease, and both no ethanol allergy symptoms. All volunteers ware no drinking within 48 hours before blood sampling. ②Experimental procedure:type Ⅱ Anerdian containing 65-75% ethanol was used as a skin disinfectant before vein haemospasia, and chlorhexidine acetate which did not contain ethanol was used as control. For the volunteers in group 1, the skins of their right elbows were cleaned three times with type Ⅱ Anerdian disinfectant without drying before immediate venipuncture; The right elbow of volunteers in group 2 use the same disinfectant clean the skin three times venipuncture after the disinfectant dried 30s. The left elbow in group 1 and 2 used ethanol-free chlorhexidine made the same disinfection and blood collection. Volunteers of group 3, the left elbow used the type Ⅱ Anerdian disinfectant cleaned the skin three times, venipuncture immediately didn’t waiting for its drying; right elbow perform the same disinfection, dry 30s before venipuncture. In the experiments, all subjects in group 1 and 2 dried non-alcohol swab was used in pressure on the wound before withdrawing the needle, group 2, completely wet cotton swab with Anerdian disinfectant was pressed on the puncture site for drawing the needle. Disposable needles were used puncture in experiments,2mL blood collected directly into K2EDTA dried vacuum anticoagulant blood collection tube, and mix, detected by the established method.(4) Measurement uncertainty analysis:Each source of uncertainty, arising from the procedure of testing was analyzed and confirmed, made a detailed study about the measurement methods, measurement systems and measurement procedures. The causal relationship structure diagram of measurement uncertainty in determination of ethanol content was constructed, based on the detected operating procedures and the formula. Draw the block diagram and measuring the flow chart of principle in measurement system or measurement methods, and calculate the corresponding measurement uncertainty. ①Sample preparation and analysis:Two hundred microliters of ethanol standard solution or blood sample and eight hundred microliters of tert-butanol(Internal standard) standard solution were placed in a 20mL headspace vial, immediately sealed with a silicone-septum cap, detected by the established method. Each alcohol concentration and blood sample make two copies of repetition, established a new ethanol standard curve, and obtained the measured blood alcohol concentration twice. ②ype A evaluation of measurement uncertainty:the concentration of 0.80 mg/mL of quality control sample was used to evaluate the measurement uncertainty of type A. Repeated the independent measured of this quality control sample 40 times under the same condition, obtained 40 parts of a detection result, find out their average value x, residual sum of squares v, experimental standard deviation s(xk) and type A evaluation of measurement uncertainty by statistical analysis.③ype B evaluation of measurement uncertainty: According to the relevant information or experience, analyzing the measured range of possible values (x-a,x+a), assumed what the probability distribution of the measured values is, k value is determined basis on the probability distribution and the required probability p value. According to the analysis, the sources of type B evaluation of measurement uncertainty were:the relative uncertainty ethanol standard solution and T-butanol introduced, relative standard uncertainty introduced by pipette and GC/MS, relative uncertainty introduced by the standard curve, peak area and lab environments, type B evaluation of measurement uncertainty u (x) was calculated by the formula: ヽombined standard uncertainty:By the standard measurement uncertainty of each input amount in a measurement model, obtained the output of standard measurement uncertainty. During the measurement of the concentration of ethanol, each input were not correlated with each other, therefore, the combined standard uncertainty is calculated as: Expanded uncertainty of the evaluation method:Expanded uncertainty U obtained by the combined standard uncertainty uc multiplying the coverage factor k. The formula is:U= kuc.(5) Drawing the quality control chart:Record each accompanying ethanol standard quality control sample which concentration was 0.80mg/mL for 100 times, with measurement concentration is 0.80mg/mL in the experiment. Calculated the mean value (x), standard deviation (s),x±3s (upper and lower control lines-UCL, LCL), x±2s (upper and lower warning line -UWL, LWL) and x±s (upper and lower guides -UAL, LAL) value. The distribution center was defined by the mean value, standard deviation indicates the unfold situation of these data. To determine the measurement results of the vertical axis, measurement procedure for the abscissa; Expected value (mean value) as the center line; ±3s for the control limits, showing measurement results of the acceptable range; ±2s for the warning limits, indicating the target region of the measurement results; ±s with the purpose of checking the range of secondary targets for the quality of the measurement results, draw out the experiment of quality control chartsResults (1) The study had established the methods of headspace gas chromatogram in detecting alcohol. After optimization method, select 45 for ethanol quantification ions,59 as tert-butanol’s quantitative ion. The retention time of ethanol and tert-butanol was 1.47min and 1.56min, respectively. General considerations equilibrium temperature and equilibration time, selected 60℃ and 20min as the headspace equilibrium temperature and time. Use ethanol concentration as x-axis, the peak area ratio of tert-butanol and ethanol quantitation ion as y-axis to draw the standard curve. The linear range of ethanol was between O.Olmg/mL and 10.00mg/mL, as the linear regression equation is.y=0.28865x-0.00304, r=0.9998. The limit of detection (LOD) and low limit of quantitation (LOQ) were 0.0006mg/mL (3S/N) and 0.001mg/mL (10S/N), respectively. The recoveries of ethanol was 99% to 112% with the valuses of RSDs(n=6) less than 5%.(2) Stability analysis of blood samples: ① Stability measurements of venous blood samples:Concentration of 22.8mg/100mL and 90.5mg/100mL venous blood which collected into the formal blood collection tube sealed, remain at room temperature (25℃) 0,2,4,6,48 and 72h, RSD were 5% and 6%, respectively, indicates that the blood sample were stable for at least 72h at room temperature. ② Stability measurements of heart blood samples:(3) The influence of the blood collection of using the disinfection on blood ethanol concentration:Blood samples were not contaminated while withdrawing the needle using a dry cotton swab to pressed on the puncture site, even a disinfectant containing ethanol was used and the skin puncture site was still wet with disinfectant. However, if an anerdian solution (containing ethanol) impregnated swab was pressed on the puncture site, there are more than four out of ten samples were contaminated even the skin puncture site was dry. The ethanol concentration of the contaminated sample was between 0.0030 and 0.0270 mg/mL.(4) Evaluation of uncertainty analysis:Using HS-GC-MS were measured blood alcohol content, the measurement uncertainty of the concentration of ethanol was came primarily from the sample determination, standard solution of the ethanol and the calibration curve. The uncertainty caused by detection equipment, internal standard, samples, etc. is very small, it can be neglected. The average of the twice measured concentration of ethanol in blood sample was 0.738mg/mL. The combined standard uncertainty was 0.042mg/mL, the expanded uncertainty was 0.084 mg/mL. k=2.(5) Draw the quality control charts:100 times QC results with the concentration of 0.08mg/mL:the mean value was 0.804 mg/mL, standard deviation was 0.045, UCL=0.938mg/mL, LCL=0.669, UWL=0.894 mg/mL, LWL=0.714, UAL=0.849 mg/mL, LAL=0.759 mg/mL. Test 10 times standard quality control samples with the obtained quality control chart, the result shows the measurement value of the standard substance substantially falls on the warning limit range, the analysis showed that the quality was normal, measurement results of the samples were reliable.Conclusion (1)The established automatic headspace-gas chromatography-mass spectrometry method makes full use of the chromatographic separation and mass spectrometry qualitative features. Ethanol concentration in the sample can be analyzed fast and accurate, no matter quantitative or qualitative. The method was simple, rapid and sensitive and good results were obtained when it was applied to determine ethanol in blood of clinical and forensic samples.(2)In the stability experiment, the venous blood taken into standard anticoagulant tube stored more stable at list 48h, can satisfied the daily examination. Heart blood samples which without anticoagulant, stable saving at room temperature (25℃) for 6 hours, refrigeration (4℃) stability for 7 days, and refrigerating (-18℃) 30 days stable. Due to the fermentation the samples alcohol concentration increased. So when the blood cannot be sent for inspecting in time and the vessels are not standardized, samples need to strictly sealed and stored in the refrigerator, frozen for the best, strictly controlled temperature storage conditions, So that it can ensure the real and effective of the results.(3) According to the hospital routine operation, the blood samples could be contaminated with ethanol while a swab containing ethanol was used to press the skin puncture site. So strengthen standardized training of the hospital blood sampling personnel, improve and standardize the operating and lancing device of suspicion drunk driving blood sampling, unify and standardize the use of blood sampling bag and blood sampling program, is important for reducing the risk of contamination and ensure justice.(4) In this section, the measurement uncertainty of the concentration of ethanol was came primarily from the sample determination, standard solution of the ethanol and the calibration curve. Therefore, the method established in this paper, can be used for evaluated the uncertainty in routine sample test results.(5) Quality control is necessary to ensure the test results accurate and reliable, experimental results shows that the quality control charts can be visually and effective control the analysis quality of blood alcohol content. And application the quality control charts to analyze the results of blood alcohol content, make effective quality control and evaluation for the measured, to provide a basis for accurate analysis of the determination of blood alcohol concentration. |
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