METHOD FOR CALCULATING DRINKING TIME

Abstract

A method for calculating drinking time includes: drawing a plurality of blood samples within 0 to 120 h upon start of drinking, testing concentrations of alcohol, EtG and EtS in the blood samples, and obtaining an average concentration ratio C.sub.EtG/C.sub.EtS; obtaining a quadratic regression equation by fitting using the average concentration ratio C.sub.EtG/C.sub.EtS as an abscissa and sampling time as an ordinate; and measuring C.sub.EtG/C.sub.EtS of blood samples under test, obtaining a relationship between the drinking time and the C.sub.EtG/C.sub.EtS based on the quadratic regression equation, and calculating the drinking time.

Claims

1. A method for calculating drinking time, comprising: drawing a plurality of blood samples within 0 to 120 h upon start of drinking, testing concentrations of EtG and EtS in the blood samples, and obtaining an average concentration ratio C.sub.EtG/C.sub.EtS of EtG to EtS; obtaining a quadratic regression equation: y=1.646x.sup.2−0.9599x+0.0878, R.sup.2=0.9904 by fitting using the average concentration ratio C.sub.EtG/C.sub.EtS as an abscissa and sampling time as an ordinate, wherein x represents the average concentration ratio C.sub.EtG/C.sub.EtS, and y represents the sampling time; and measuring C.sub.EtG/C.sub.EtS of blood samples under test, obtaining a relationship between the drinking time and the C.sub.EtG/C.sub.EtS based on the quadratic regression equation, and calculating the drinking time.

2. The method for calculating drinking time according to claim 1, wherein a blood alcohol concentration upon drinking is in the range of 0.22 to 0.66 mg/m.

3. The method for calculating drinking time according to claim 2, wherein an alcohol intake amount is 0.72 g/kg.

4. The method for calculating drinking time according to claim 1, wherein sampling intervals of the blood samples used for obtaining the quadratic regression equation are at 0 h, 0.5 h, 2 h, 3 h, 5 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 120 h respectively.

5. The method for calculating drinking time according to claim 1, wherein the concentrations of EtG and EtS in the blood samples are tested by: S1. pre-treating the blood samples transferring the blood samples into centrifuge tubes added with internal standards EtG-D.sub.5 and EtS-D.sub.5, adding 80% of acetonitrile in methanol, precipitating and centrifuging at 0 DEG C., transferring supernatant, drying, re-dissolving with 5% of acetonitrile in water, centrifuging again, and taking the supernatant to obtain the blood samples under test; and S2. measuring concentrations of EtG and EtS by liquid chromatography-tandem mass spectrometry for the blood samples under test in S1.

6. The method for calculating drinking time according to claim 5, wherein in S2, a separation condition for liquid chromatography comprises the following parameters: chromatographic column: an Inertsil ODS-3 column, 2.1 mm×100 mm, 3 μm; and column temperature: 35 DEG C.; and in an elution system, mobile phase A: 0.1% of formic acid in water, mobile phase B: 0.1% of formic acid in acetonitrile; flow rate: 0.2 mL/min; and gradient elution procedures: 0 to 2 min, a volume ratio of the mobile phase A to the mobile phase B is 95:5; 2 to 6 min, a volume ratio of the mobile phase A to the mobile phase B is 10:90; 6 to 8 min, a volume ratio of the mobile phase A to the mobile phase B is 10:90; and 8.5 to 14 min, a volume ratio of the mobile phase A to the mobile phase B is 95:5.

7. The method for calculating drinking time according to claim 5, wherein in S2, a test condition for a mass spectrum comprises the following parameters: electrospray ionization in a negative mode; and voltage of ion spray: −4000 V, and temperature: 500 DEG C.

8. The method for calculating drinking time according to claim 5, wherein in S 1, a concentration of the internal standard EtG-D.sub.5 is 1 μg/mL, and a concentration of the internal standard EtS-D.sub.5 is 1 μg/mL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 shows average concentration-time curves of alcohol, EtG and EtS in blood;

[0036] FIG. 2 shows liquid chromatography-mass spectrometry (LC-MS) chromatograms (500 ng/mL) of EtG, EtS, and internal standard EtG-D.sub.5 and EtS-D.sub.5;

[0037] FIG. 3 shows an LC-MS chromatogram of a blood-blank sample; and

[0038] FIG. 4 shows LC-MS chromatograms (500 ng/mL) of a blood-blank sample added with EtG, EtS, and internal standard EtG-D.sub.5 and EtS-D.sub.5.

DETAILED DESCRIPTION

[0039] For clearer description of the objects, technical solutions, and advantages of the present disclosure, the present disclosure is further described in detail hereinafter with reference to the accompanying drawings and some exemplary embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present disclosure and should not be deemed as limiting the scope of the present disclosure.

[0040] Based on the embodiments of the present disclosure, all other embodiments obtained by those ordinary skilled in the art without creative efforts should fall within the scope of protection of the present disclosure.

[0041] The drinking time described in the present disclosure refers to the time from the start of drinking when the samples are taken for testing.

[0042] Unless otherwise specified, the experimental methods in the embodiments mentioned below are conventional methods, and the reagents and materials are available commercially.

Example 1

[0043] 1. Materials and methods

[0044] 1.1. Chemicals and reagents

TABLE-US-00001 Alcohol (10 mg/mL) Accustandard, USA; Tert-Butanol (AR, ≥99.0%) Aladdin, Shanghai; EtG (100 mg/mL) Cerilliant, USA; Internal standard EtG-D.sub.5 (IS; 1 μg/mL) Cerilliant, USA; Internal standard EtS-D.sub.5 (IS; 1 μg/mL) Cerilliant, USA; EtS-Na (98%) TSI, Japan; Methanol (HPLC grade) Merke, USA; Acetonitrile (HPLC grade) Merke, USA; Formic acid (LC/MS grade) Bailingway, China; and Ultrapure water Milli-Q Ultrapure Water System, USA

[0045] 1.2. Participants and experimental methods

[0046] Approved by the Medical Ethics Committee of Shanxi Medical University (2018LL349), a total of 26 adults including 14 men and 12 women are recruited by the team to participate in the study. All participants have no histories of physical or mental illness, and drinking or medication, of which, a median age is 24.5 years old (in the range of 22 to 27 years old), and a mean body mass index is 20.9 kg/m.sup.2 (in the range of 16.8 kg/m.sup.2 to 34.6 kg/m.sup.2).

[0047] Participants signed informed consent forms prior to the start of the study. For safety, all participants were observed in a school hospital for at least 24 h upon drinking, and were medically evaluated accordingly during drinking and 3 days upon drinking.

[0048] Upon a 12-h fast, the participants drank (Fenjiu, with an alcohol content of 40%) with food within 30 minutes according to a dose standard of 0.72 g/kg (which is proportional to weights of the participants), and 5 mL of blood was drawn through indwelling catheters in median cubital veins before (0 h) drinking and 0.5 h, 1.5 h, 2 h, 3 h, 5 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 120 h upon drinking respectively, as blood samples under test. All the blood samples were stored at −20 DEG C. until the end of the analysis.

[0049] 1.3. Samples preparation

[0050] 1.3.1. Preparation of blood samples under test containing internal standard tert-butanol

[0051] Alcohol contents in the blood samples were measured using a headspace gas chromatography internal standard method with tert-butanol as an internal standard. 1 mL of blood and 1 mL of tert-butanol (IS, 87 mg/mL) were added to a headspace vial, diluted with 3 mL of ultrapure water, mixed in a sealed condition, and then analyzed by the headspace gas chromatography.

[0052] 1.3.2. Preparation of blood samples under test containing internal standards EtG-D.sub.5 and EtS-D.sub.5

[0053] Metabolites EtG and EtS in blood samples were measured by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) with EtG-D.sub.5 and EtS-D.sub.5 as internal standards. The internal standards EtG-D.sub.5 (IS, 1 μg/mL) and EtS-D.sub.5 (IS, 1 μg/mL) were taken 100 μL separately, and mixed well to obtain mixed internal standards; 100 μL of blood was taken and 100 μL of mixed internal standard was added to improve the identification and quantification of the metabolites (EtG and EtS). Then 800 μL of 80% acetonitrile in methanol was added, and the mixture is precipitated at 0 DEG C. for 10min. After that, centrifugation was performed at 13000 rpm for 5 min, supernatant was taken out, and dried, by blowing, with nitrogen at 35 DEG C., and then re-dissolved with 400 μL of 5% acetonitrile in water and centrifuged again at 13000 rpm for 5 min. 3 μL of the supernatant was taken and injected into the LC-MS/MS for analysis, as shown in FIGS. 2 to 4.

[0054] 1.4 Mass spectrometry analysis

[0055] Chromatograph separation was performed via an LC-20 A system. Conditions of the chromatograph were as follows:

[0056] chromatographic column: Inertsil ODS-3 column (2.1 mm×100 mm, 3 μm; Shimadzu, Japan) , and column temperature: 35 DEG C.

[0057] Mobile phases: mobile phase A (0.1% of formic acid in ultrapure water) and mobile phase B (0.1% of formic acid in acetonitrile); gradient elution (see Table 1); flow rate: 0.2 mL/min; total time of elution: 14.0 min; and injection volume: 5 μL.

TABLE-US-00002 TABLE 1 Gradient elution conditions Time/min A/% B/%   0-2.0  95 5 2.0-6.0  10 90 6.0-8.0  10 90 8.0-8.5  95 5 8.5-14.0 95 5

[0058] Targeted substances were tested by a tandom mass spectrometer (TRAP4,000, Sciex, AB). The specific conditions were as follows:

[0059] Ion source: electron spray ionization (ESI); voltage of ion spray: −4000 V, and temperature: 500 DEG C.; curtain gas, nebulizer (Gas 1), and heating auxiliary gas (Gas 2): 40 psi, 50 psi, and 35 psi respectively.

[0060] Scanning mode: anions+multiple reaction monitoring (MRM).

[0061] Other specific MRM parameters for each analyte are shown in Table 2.

TABLE-US-00003 TABLE 2 Characteristic ion pairs and mass spectrometric data for each analyte Targeted Qualitative ions Quantitative Declustering Collision substances (m/z) ions (m/z) potential (V) energy (V) EtS 125.0/80.0 125.0/97.0 46 45 125.0/97.0a 21 EtG 221.1/75.0a 221.1/75.0 63 22 221.1/85.0 23 EtG-D.sub.5 226.1/75.0a 226.1/75.0 63 23 226.1/85.0 26 EtS-D.sub.5 130.0/80.0a 130.0/80.0 46 46 130.0/97.9 25 Note: a represents a quantitative ion pair.

[0062] 1.5. Estimation of the last drinking time

[0063] The last drinking time was estimated based on a relationship between the average concentration ratio C.sub.EtG/C.sub.EtS of EtG to EtS and the sampling time, and an error between observed time and actual time was calculated according to the following formula:

Error=(observed value−actual value)/actual value)*100%.

[0064] In the formula, the observed value represents theoretical observed time, that is, the estimated time of the last drinking; and the actual value represents the actual time, that is, actual sampling time since the last drinking.

[0065] 1.6. Statistics

[0066] The pharmacokinetic parameters were calculated by a non-compartmental model using a DAS 3.0 software. All data was summarized using descriptive statistics. Some key data including arithmetic mean values and standard deviations of targeted substance concentrations, detection time points, pharmacokinetic parameters and other results were provided. All statistical analyses were performed using version 13.0 of an IBM SPSS® software (SPSS Inc., Chicago, IL, USA).

[0067] 2. Results

[0068] 2.1. Verification of the method

[0069] Limit of detection (LOD) and limit of quantitation (LOQ) for the EtG and EtS in blood samples were 0.02 μg/mL and 0.05 μg/mL respectively. Residue obtained from previous treatment was resolved with 100 μL of 5% acetonitrile aqueous solution to quantify concentrations below LOQ.

TABLE-US-00004 TABLE 3 Linear ranges and LOD of EtG and EtS in blood Targeted Linear LOD LOQ sub- ranges (μg/ (μg/ stances (μg/mL) Linear equation R.sup.2 Weight mL) mL) EtG 0.05 − 5.0 y = 1.7411x + 0.0036 0.9999 Non 0.02 0.05 EtS 0.05 − 5.0 Y = 1.803x − 0.0279 0.9997 Non 0.02 0.05

TABLE-US-00005 TABLE 4 Precision, recovery and matrix effects of EtG and EtS in blood Targeted Con- Within-batch Between-batch Re- Matrix sub- centration precision precision covery effect stances (μg/mL) (%) (%) (%) (%) EtG 0.05 5.2 4.8 70.1 15.4 0.5 4.8 2.4 66.9 11.5 5.0 4.7 2.0 66.1 11.9 EtS 0.05 4.0 5.7 85.1 3.9 0.5 4.7 2.0 82.0 1.0 5.0 2.5 6.1 88.6 0.9

[0070] As shown in Tables 3 to 4, all analytes including alcohol, EtG, EtS, EtG-D.sub.5 and EtS-D.sub.5 are well separated and no endogenous peaks are eluted by the analytes, such that the method is verified fully. 2.2. Estimation of the last drinking time

TABLE-US-00006 TABLE 5 Average concentrations (x ± S (min − max), n = 26) of alcohol and metabolites thereof in human blood Time BAC (mg/mL) EtG (μg/mL) EtS (μg/mL) 0 — — — 0.5 h 0.34 ± 0.10 0.05 ± 0.02 0.06 ± 0.02 (0.19 − 0.61) (0.02 − 0.08) (0.03 − 0.09) 1.5 h 0.41 ± 0.11 0.14 ± 0.04 0.11 ± 0.02 (0.22 − 0.66) (0.06 − 0.24) (0.06 − 0.16)   2 h 0.41 ± 0.12 0.20 ± 0.06 0.14 ± 0.03 (0.14 − 0.60) (0.10 − 0.34) (0.07 − 0.20)   3 h 0.36 ± 0.14 0.27 ± 0.09 0.16 ± 0.04 (0.07 − 0.63) (0.13 − 0.47) (0.07 − 0.25)   5 h 0.17 ± 0.10 0.29 ± 0.12 0.14 ± 0.05 (0.00 − 0.37) (0.09 − 0.53) (0.03 − 0.24)   8 h 0.03 ± 0.04 0.14 ± 0.08 0.06 ± 0.03 (0.00 − 0.14) (0.04 − 0.30) (0.01 − 0.11)  12 h — 0.04 ± 0.03 0.02 ± 0.01 (0.01 − 0.12) (0.00 − 0.04) Note: “—” represents “not detected”; BAC represents blood alcohol concentration; and all values are accurate to two decimal places. The alcohol, EtG and EtS are not detected at 24 h, 36 h, 48 h, and 120 h upon drinking. The intervals for collecting blood samples are selected from 0 to 120 h, which is based on the fact that a detection window period of non-oxidative metabolites of alcohol are longer than that of elementary bodies of alcohol reported in the literature. However, in the detection process of the embodiments of the present disclosure, it is found that the individual targeted substance is undetectable after 24 h.

[0071] According to the average concentration of the EtG and EtS in blood samples as shown in Table 5, the average concentration ratio C.sub.EtG/C.sub.EtS of EtG to EtS is calculated, and the relationship between the ratio after a single oral dose and the last time of use is analyzed, showing that a quadratic regression equation y=1.646x.sup.2−0.9599x+0.0878, R.sup.2=0.9904, is obtained by using the average concentration ratio C.sub.EtG/C.sub.EtS as an abscissa and the sampling time as an ordinate. In the formula, x represents the average concentration ratio C.sub.EtG/C.sub.EtS, and y represents the sampling time.

TABLE-US-00007 TABLE 6 Errors between the time deduced from a quadratic function and the actual last drinking time C.sub.EtG/ Observed value Actual Error C.sub.Ets (h) (CI) value (h) (%) — 0.00 0.00 0.00 0.79 0.35 (0.27 − 0.63) 0.50 29.05 1.22 1.36 (1.16 − 2.04) 1.50 9.06 1.42 2.03 (1.64 − 3.02) 2.00 1.41 1.65 2.99 (2.68 − 4.86) 3.00 0.18 2.13 5.53 (5.04 − 8.43) 5.00 10.54 2.45 7.64 (6.57 − 11.52) 8.00 4.46 CI represents a confidence interval (95%).

[0072] As shown in Table 6, the concentration ratio of EtG to EtS is substituted into the regression equation (y=1.646x.sup.2−0.9599×+0.0878, in which x represents ratio, y represents time and R.sup.2=0.9904) to calculate the observed value of the drinking time. The error between the observed value and the actual value within 8 h is obtained using the error calculation formula (error=(observed value−actual value)/actual value)* 100%), and the errors are basically less than 10%.

[0073] 2.3. Pharmacokinetic analysis

[0074] The average concentrations of the alcohol and metabolites thereof in human blood at each time point are shown in Table 5, and LOD data for the alcohol and metabolites thereof are shown in Table 7.

TABLE-US-00008 TABLE 7 LOD data (x ± S (min − max), n = 26) for alcohol and metabolites thereof in human blood Targeted substances Alcohol EtG EtS LOD (h) 5.81 ± 1.74 22.15 ± 4.42 16.92 ± 6.23 (3.00 − 8.00) (12.00 − 24.00) (8.00 − 24.00)

[0075] The result shows that after drinking 0.72 g alcohol/kg, the average blood alcohol concentration of the participants reaches 0.41±0.11 mg/mL 1.5 h latter, and then gradually decreases, with a detection window time (maximum observed value) of 3 to 8 h.

[0076] The metabolites EtG (0.29±0.12 μg/mL) and EtS (0.16±0.04 μg/mL) reach peak values at 5 h and 3 h respectively.

[0077] In addition, as shown in FIG. 1, in the study process, it is found that the concentration of EtG is consistently higher than that of EtS.

[0078] Based on the non-compartmental model, after the participants drink 0.72 g alcohol/kg, the pharmacokinetic parameters for the alcohol in blood as well as the metabolites EtG and EtS are calculated, to obtain a pharmacokinetic model, and the result is shown in Table 8.

TABLE-US-00009 TABLE 8 Pharmacokinetic parameters (x ± S, min − max, n = 26) for alcohol and metabolites thereof in human blood Parameters Samples containing alcohol EtG EtS AUC (0-t) 1,715.23 ± 626.72 1.99 ± 0.78 1.04 ± 0.34 (mg/L*) (505.00 − 2,914.00) (0.76 − 3.55) (0.41 − 1.75) t1/2z (h) 0.241 ± 1.09 2.56 ± 0.89 2.04 ± 0.76 (0.30 − 4.23) (1.03 − 4.94) (1.11 − 3.32) T.sub.max (h) 2.02 ± 0.54 4.12 ± 1.07 3.02 ± 0.70 (1.50 − 3.00) .00 (2 − 5.00) (1.50 − 5.00) C.sub.max (mg/L) 441.65 ± 113.86 0.31 ± 0.11 0.17 ± 0.04 (238.20 − 656.00) (0.13 − 0.53) (0.08 − 0.28) Vz/F (L/kg) 0.69 ± 0.49 — — (0.11 − 1.76) Clz/F (L/h) 0.49 ± 0.33 — — (0.17 − 0.43) Note: AUC (0-t) represents an area under the curve; t1/2z represents a half-life period; T.sub.max represents time to peak; C.sub.max represents a peak concentration; Vz/F represents apparent volume of distribution; and Clz/F represents a clearance rate.

[0079] The result shows that the peak concentration (441.65±113.86 mg/L (0.44±0.11 mg/mL)) of the alcohol is reached at 2.02±0.54 h. The peak concentrations (0.31±0.11 mg/L and 0.17±0.04 mg/L) of the metabolites are reached at 4.12±1.07 h and 3.02±0.70 h. T1/2z of alcohol, EtG and EtS are at 1±1.09 h, 2.56±0.89 h and 2.04±0.76 h. Clz/F of alcohol is at 0.49±0.33 L/h. However, due to in-vivo doses of metabolites (EtG and EtS) of alcohol are unknown, the Vz/F and CLz/F for both cannot be accurately calculated.

[0080] 3. Discussion

[0081] According to the present disclosure, the drinking time is inferred, mainly based on the pharmacokinetic study, using a variation pattern of the average concentration ratio between non-oxidative metabolites of alcohol over time. Specifically, a regression equation is established based on the average concentration ratio of EtG to EtS in blood and the drinking time, and thus a regression equation y=1.646x.sup.2−0.9599x+0.0878, R2=0.9904 in a 0-8 h window period is obtained, which indicates that the average concentration ratio of EtG to EtS in blood has a good correlation with the time of using alcohol. The average concentration ratio of EtG to EtS is substituted into this equation to calculate a theoretical value of the drinking time using an inverse method. Meanwhile, inference errors are calculated using the formula “(theoretical value−measured value)/actual drinking time,” revealing that the errors are basically less than 10%.

[0082] The LOD and LOQ of the non-oxidative metabolites (EtG and EtS) of alcohol in blood samples are 0.02 μg/mL and 0.05 μg/mL respectively, indicating that the method of the present disclosure can effectively quantify the EtG and EtS with lower concentrations in blood. The blood alcohol concentration (BAC) of 0.72 g/kg alcohol dose in the embodiments of the present disclosure is in the range of 0.22 to 0.66 mg/mL, which is similar to the existing BAC standard for determining drunk driving (>0.2 mg/mL), indicating that the method of the embodiments of the present disclosure is applicable to monitoring of most drunk driving cases in China.

[0083] According to the present disclosure, based on the non-compartmental model, the pharmacokinetic parameters of the alcohol, EtG and EtS in blood are calculated, which indicates that the peak concentration C.sub.max (441.65±113.86 mg/L (0.44±0.11 mg/mL)) of the alcohol is reached at 2.02±0.54 h. Compared with previous studies, an absorption phase of the alcohol obtained by the embodiments of the present disclosure is longer, that is, the absorption is slower. Furthermore, it is found that alcohol could be detected in the participants' blood within 3 to 8 h and the average elimination half life of the alcohol is at 1.24±1.09 h (0.30 to 4.23 h).

[0084] According to the present disclosure, based on the non-compartmental model, the pharmacokinetic parameters of the alcohol, EtG and EtS in blood are calculated, which indicates that the metabolites EtG and EtS have longer detection window periods, and confirms that metabolism velocity of EtG is slower than that of alcohol, and the elimination half life of EtG in blood is at 2.56±0.89 h. EtS is another non-oxidative metabolite of alcohol metabolism with a concentration-time curve similar to that of EtG. In the studies, at a dose of 0.72 g/kg, the peak concentration C.sub.max of EtS is 0.17 μg/mL (in the range of 0.08 μg/mL to 0.28 μg/mL), and the peak time T.sub.max is at 3.02 h. In addition, it is also found that the detection window period and peak concentration C.sub.max of EtS are significantly lower than those of EtG. However, EtS is more stable and insensitive to bacteria. Accordingly, the EtS can provide supplementary data for identifying alcohol intake.

[0085] In conclusion, according to the studies of the present disclosure, an idea and method for inferring the drinking time using the average concentration ratio EtG/EtS, which, after further verification, are expected to provide a useful analytical monitoring tool for drunk-driving identification and related inference of the drinking time in China. Moreover, pharmacokinetics of EtG and EtS in blood of Chinese population are further studied, and the pharmacokinetic parameters for both are obtained. The sensitive LC-MS/MS method developed and verified in the embodiments of the present disclosure can be applied to drunk-driving and other forensic cases involving alcohol. The long detection window periods of EtG and EtS support the EtG and EtS as useful markers for detecting alcohol consumption.

[0086] The above described are merely the preferred embodiments of the present disclosure, not used for limiting the present disclosure. Any modifications, equivalent replacements, improvements and the like made within the spirit and principles of the present disclosure should be included in the scope of protection of the present disclosure.