Abstract
Due to the forensic aspects of drinking and exposure to toxic substances, more sophisticated quantitative technology is needed to quantify the concentration of ethyl alcohol and acetaldehyde in the blood. In this study, we developed a headspace gas chromatography tandem mass spectrometry method that could simultaneously detect ethyl alcohol and acetaldehyde in human plasma. Through a simple preparation process, ethyl alcohol and acetaldehyde were quickly detected within 4 min, and a lower limit of quantification (LLOQ) (20 and 0.2 µg/mL) was obtained; these results confirmed the suitability of the system. According to Food and Drug Administration guidelines, the linearity (correlation coefficient > 0.9996), intraday and intraday accuracy, precision, and both short- and long-term stability and freeze–thaw stability satisfied the evaluation criteria (within 100.0 ± 15.0% and 20.0% of the LLOQ). Carryover and batch size assessment for the evaluation of the sample influence during analysis also satisfied the evaluation criteria. A valid method was applied to the pharmacokinetics study, and the plasma from 43 subjects after oral administration of the placebo or HK-GCM-H01 was analyzed. The developed analysis method for ethyl alcohol and acetaldehyde in blood could be used in various fields, such as forensics and those requiring precise quantification.
Keywords: Ethyl Alcohol, Acetaldehyde, Gas Chromatography-Mass Spectrometry, Validation Study, Pharmacokinetics
INTRODUCTION
The blood alcohol concentration (BAC) refers to the concentration of alcohol in the blood as a percentage and is usually used as a medical and legal measure of alcoholism. The Enforcement Decree of the Road Traffic Act in the Republic of Korea stipulates that blood needs to be collected and that BAC needs to be analyzed to crack down on drunk driving in the case of traffic violations. If drunk driving is suspected, the BAC at the time of the accident can be estimated using the formula presented by Widmark [1]; this formula is based on the amount and type of alcohol consumed by the driver before the accident, weight, sex, etc. Currently, many modeling methods for calculating the BAC have been developed [2]; however, without analyzing the BAC, no reliable alternative presently exists. Additionally, excessive drinking is especially damaging to the liver because the main pathway for the elimination of alcohol from the body is through its oxidation to acetaldehyde by alcohol dehydrogenase and cytochrome P450 2E1 in the liver; this pathway eliminates 90% of the alcohol [3,4,5]. Then, acetaldehyde is quickly broken down into a less toxic compound called acetate by aldehyde dehydrogenase. Acetaldehyde is a byproduct of ethyl alcohol, is a highly toxic substance and a carcinogen in the body [6] and is implicated in aging and age-related diseases such as cardiovascular and neurological diseases [7,8]. Although acetaldehyde is generally short-lived, it is known to cause headaches during hangover [9]. Recently, substances have been developed that eliminate the side effects of hangovers by blocking the metabolic process of acetaldehyde [10]. Similarly, identifying the blood acetaldehyde concentration (BAcHC) and BAC can be helpful in scientific and commercial areas.
Currently, many methods have been developed to quantify BAC and BAcHC, and the instruments used are diverse; they include liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) [11,12,13,14,15,16,17,18]. Brien and Loomis’s study (1978) [17] simultaneously analyzed alcohol and acetaldehyde in blood using GC and performed a pharmacokinetic (PK) study on a male volunteer. Although the study was based on only one subject, the results were relatively clear for the technology available at that time. Research was subsequently conducted by McCarver-May and Durisin [15] in 1997 using gas chromatography (GC) with headspace to analyze acetaldehyde, which is highly volatile in the body. In the 21st century, many headspace gas injection methods, coupled with mass spectrometry, became popular for BAC quantification. In 2013, Cordell developed an analysis method to detect ethyl alcohol and other volatile compounds using headspace gas chromatography mass spectrometry (HS-GC-MS) and attempted to analyze the blood of neonatal patients [19]. The analytical methods had low limits of detection and limits of quantification (LOQs), and acceptable accuracy and precision; however, the methods were not perfect because the interday variability of acetaldehyde, which is a primary metabolite of ethyl alcohol, was high at > 15%. In another study, a simple ethyl alcohol detection method using HS-GC-MS in whole blood was developed, but this method required a rather long analysis time, with a total run time of 14 minutes [20]. In addition to the importance of acetaldehyde as alcohol consumption increases, a developed HS-GC-MS method can reliably determine the concentration of acetaldehyde along with ethyl alcohol, but this method focuses on the nerves through the brain rather than the blood [14]. To date, most HS-GC-MS detection methods for ethyl alcohol and acetaldehyde have excellent sensitivity and are valid however, no studies have yet applied this method to pharmacokinetics (PK) research.
In this study, we aimed to develop an analytical method with high sensitivity and excellent resolution for simultaneously detecting ethyl alcohol and its metabolite, acetaldehyde, using headspace gas chromatography–tandem mass spectrometry (HS-GC-MS/MS). Additionally, we applied PK studies on ethyl alcohol and acetaldehyde in human blood using headspace, as a fast, robust analytical method with simple pretreatment.
METHODS
Chemicals
Ethyl alcohol (> 99.9%), acetaldehyde (≥ 99.8%), tert-butanol (≥ 99.0%), thiourea and sodium fluoride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water (18.2 MΩ∙cm) was obtained using a Merck Milli-Q apparatus from Millipore (Milford, MA, USA). HK-GCM-H01, manufactured as a clinical trial product, consists of 77 types of hangover relief ingredients.
Clinical study design
The clinical study followed a randomized, double-blinded, placebo-controlled crossover design to limit intrasubject variability (Supplementary Fig. 1). Fifty Korean male volunteers who voluntarily agreed to participate and signed an informed consent form were recruited using the following inclusion criteria: age, 26.96 ± 4.54 years; body mass index, 22.95 ± 1.32 kg/m2. The participants were hospitalized on the afternoon of the day before the first intake day (1st period, Day −1) and were provided with the same prepared dinner while fasting for more than 8 hours. Then, the participants were randomly assigned to receive one of the following 2 groups (Reference and Test). On the first day of intake (1st period, Day 1), blood was collected before the start of alcohol intake (0 hour samples), and meals served at 2-minute intervals were completed according to the prescribed order. Approximately 2 hours after the start of the meal, the placebo or HK-GCM-H01 was consumed at 2-minute intervals in the order of participation number in accordance with the randomization results. Thirty minutes after intake, alcohol (0.78 g/kg b.w.) was consumed within 15 minutes at 2-minute intervals, and drinking water was restricted for 2 hours. Then, scheduled pharmacokinetic blood collection was performed, and the same amount of water was allowed to be consumed 2 hours after the start of alcohol consumption. Lunch was provided 4 hours later. Scheduled blood collection was performed until 15 hours later, and the participants were discharged the next day (1st period, Day 2). There was a wash-out period of 8 days after the first intake date, and the second period test (2nd period, Day 1) was conducted on the same schedule as the first period test, including hospitalization the day before the second period intake day (2nd period, Day −1). When the final clinical trial was completed, 43 out of 50 subjects completed the human application test. The study protocol was approved by the Institutional Review Board (IRB) of Chungbuk National University Hospital (IRB No. 2023-09-021). All participants provided written informed consent before their inclusion.
Standard preparation
Ethyl alcohol and acetaldehyde were weighed to determine their density and purity and were dissolved in distilled water to concentrations of 40,000 and 40 µg/mL, respectively. The calibration curves were prepared using seven concentrations serially diluted from each stock solution for quantitative analysis. The quality control (QC) samples for each standard were utilized at four different concentrations containing the lower limit of quantification (LLOQ) (LLOQ, low, medium and high) made from stocks of each standard. The detailed concentration points of the calibration curve and QC samples are listed in Table 1. All stock and working solutions were diluted with distilled water. The stock solution was subsequently stored under refrigeration and protected from light, and each working solution was used immediately after preparation.
Table 1. Final concentration of ethyl alcohol and acetaldehyde for calibration curve and QCs.
| Sample name | Concentration (µg/mL) | ||
|---|---|---|---|
| Ethyl alcohol | Acetaldehyde | ||
| Calibration curve | |||
| Blank | 0 | 0 | |
| StD1 | 20 | 0.2 | |
| StD2 | 50 | 0.5 | |
| StD3 | 100 | 1 | |
| StD4 | 200 | 2 | |
| StD5 | 500 | 5 | |
| StD6 | 1,000 | 10 | |
| StD7 | 2,000 | 20 | |
| QC | |||
| LLOQ | 20 | 0.2 | |
| QC-Low | 60 | 0.6 | |
| QC-Medium | 400 | 4 | |
| QC-High | 1,600 | 16 | |
QC, quality control; StD, standard; LLOQ, lower limit of quantification.
Blood sample preparation
Blood preparation was performed for quantitative analysis of each product. After separating the plasma from the blood through centrifugation, 0.5% sodium fluoride was added. Then, 200 µL of 200 mM thiourea solution was added to 200 µL of each plasma sample to achieve a 1:1 ratio in the headspace vial, and the mixture was mixed gently by hand. Afterward, 200 µL of distilled water containing 50 µg/mL tert-butanol (internal standard [IS]) was added, and the mixture was vortexed. Finally, the mixed samples were injected into the HS-GC-MS/MS system.
HS-GC-MS/MS
An Agilent 8890 series gas chromatography system (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 8697A headspace (Agilent Technologies) and an Agilent 7010C series mass spectrometry system (Agilent Technologies) were used. The oven and loop temperatures of the headspace were 70°C and 80°C, respectively, and the GC cycle time was 7 minutes. The vial equilibration time was 7 minutes, and the injection duration time was 0.5 minutes. The samples were injected into an Agilent DB-BAC2 UI (30 m × 0.32 mm, 1.2 µm column with an inlet temperature of 150°C). The column flow rate was 2 mL/min using helium carrier gas, and the split ratio was 300:1. The oven temperature was regulated by the initial temperature of 40°C for 4 minutes isothermally. The ionization method was electron impact, and the source and quadruple temperatures were 230°C and 150°C, respectively. All ions were detected under selected ion monitoring (SIM) mode, and the analytical parameters of each standard sample containing an IS were as follows: ethyl alcohol (m/z 31.0), acetaldehyde (m/z 29.0) and tert-butanol (m/z 41.0).
Method validation
Linearity
A calibration curve was constructed based on the peak area ratio of the analyte to the IS versus the concentration of each standard sample, and y = ax + b and 1/x2 was used for linear regression and weighting factor, respectively. Blank samples were not used when the calibration curve was created. The concentrations of ethyl alcohol and acetaldehyde were obtained by back-calculation from the calibration curve. For the validation of linearity, the correlation coefficient (r) of the calibration curve was 0.9900 or greater, and the accuracy of the inversion concentration for each standard for the calibration curve was within 100.0 ± 15%.
Accuracy and precision
The interday accuracy and precision were determined using QC samples collected at least three times over two or more different days, and the intraday accuracy and precision were determined by analyzing five replicates of the QC samples collected on the same day. The concentration from each trial was calculated from the calibration curve of the same analytical batch, and the accuracy and precision were evaluated from the calculated concentration. The accuracy (%) was calculated by dividing the measured average concentration by the nominal concentration, and the precision (%) was calculated by dividing the standard deviation by the average concentration. The acceptable limits of interday and intraday accuracy and precision were within 100.0 ± 15% bias.
Stability
Among the four QC concentrations, only the QC-Low and QC-High samples were used for validation of stability, and each concentration was analyzed in triplicate. The concentrations of the stability samples were calculated from the calibration curve of the same analytical batch, and the accuracy and precision were evaluated from the calculated concentration. For freeze-thaw stability, the stable samples were stored in cryogenic freezers (−85°C to −65°C) for more than 24 hours and then thawed at room temperature. Once completely thawed, the samples were again frozen under the same conditions for more than 12 hours. The freezing and thawing process was repeated three times; afterward, the samples were injected into the analytical instrument. To determine the stability according to the storage period, the stability was measured by splitting the samples between two storage periods. In each case, the QC samples were left at room temperature for less than 24 hours (short-term stability), and the QC samples were stored in cryogenic freezers (−85°C to −65°C) for less than 10 days before injection (long-term stability). For validation of stability, the sample was considered stable if the average concentration accuracy of the calculated concentration of the stability sample processed under the corresponding conditions for each stability validation was within 100.0 ± 15.0% and the precision was 15.0% or less.
Carryover
For validation of carryover, the highest concentration of the calibration curve (standard [StD]7) was initially injected and run through the system; immediately after, a blank sample without a standard was injected three times to evaluate the presence and influence of standard materials and IS peaks. The carryover (%) was calculated by dividing the peak area of the blank samples by the peak area of the LLOQ sample. For the validation of carryover using the blank samples, the peak area of the standard and IS should be less than 20.0% of the peak area of the standard and less than 5.0% of the peak area of the IS that is detected at the lowest limit of the quantification sample.
Batch size assessment
All QC samples were pretreated to the target number and then continuously injected into the HS-GC-MS/MS system. At the time of injection, one set was composed of 5 doses for each concentration in the order of each QC sample (QC-Low, QC-Medium and QC-High). The batch size was determined as the number of samples and included a set with an accuracy of less than 100.0 ± 15.0% and a precision of less than 15% for the average concentration of the calculated concentration for each concentration, the blank (noting all), the zero sample (only the IS), and the calibration curve samples.
All validation was based on the Food and Drug Administration (FDA) guidance for industry: bioanalytical method validation [21] and Korean Food and Drug Administration (KFDA) guidance for industry: bioanalytical method validation [22].
Data analysis
Data were processed via Mass-Hunter Quantitative Analysis 12.0 (Agilent Technologies) using the operating program of the GC-MS/MS, and the results were obtained.
PK study
For PK, the parameter values were calculated for each participant, and then the average and standard deviation for each intake group were calculated. Descriptive statistics were calculated for the pharmacokinetic evaluation variables and included Cmax, AUC0-15h, Tmax, and t1/2 of alcohol and acetaldehyde; these variables are the evaluation variables of PK, and comparisons between the groups were performed. The PK data were processed with Phoenix WinNonlin® Version 8.3 (Certara, Princeton, NJ, USA).
RESULTS
Development of the analytical method
As shown in Fig. 1, ethyl alcohol and acetaldehyde were detected using HS-GC-MS/MS at 2.051 and 1.535 minutes. Also, the tert-butanol was used as an IS and had a retention time of 2.721 minutes. The mass to charge of ethyl alcohol, acetaldehyde and tert-butanol were quantified at m/z 31.0, 29.0 and 41.0, respectively (Fig. 1).
Figure 1. The selected ion monitoring chromatogram of ethyl alcohol, acetaldehyde and tert-butanol in headspace gas chromatography-tandem mass spectrometry analysis. Each chromatogram in marked with the detected concentration of QCs and the detected m/z was indicated of ethyl alcohol (A) and acetaldehyde (B). (C) Chromatogram and mass spectrum of tert-butanol as Internal standard.
QC, quality control.
Validation of the analytical method
In this study, we developed an analytical method to detect ethyl alcohol and acetaldehyde in human plasma using HS-GC-MS/MS and measured their concentrations using tert-butanol as an IS. The concentrations of the QC samples for each validation are listed in Table 1.
Linearity
In the quantitative range of 20 µg/mL to 2,000 µg/mL for evaluating the linearity of ethyl alcohol, the least-squares regression (r2) of the calibration curve was 0.9996; these results satisfied the acceptance criteria (least-squares regression 0.9900 or more) (Fig. 2). The accuracies of the calculated concentration of the calibration curve samples excluding the lowest limit of quantitation were 98.2% to 102.9%, and the accuracy of the LLOQ sample was 100.8%; this was determined by the satisfaction of the acceptance criteria (accuracy of 100.0 ± 15%, with the LLOQ sample of 100.0 ± 20%). In the quantitative range of 0.2 µg/mL to 20 µg/mL for evaluating the linearity of acetaldehyde, the least-squares regression (r2) of the calibration curve was 0.9998; this satisfied the acceptance criteria. The accuracy of the calculated concentration of the calibration curve samples was 97.8%, and the accuracy of the LLOQ sample was 100.0%; these results also satisfied the acceptance criteria. The detailed results from the calibration curve are provided in Fig. 2 and Table 2.
Figure 2. Calibration curves of ethyl alcohol (A) and acetaldehyde (B).
Table 2. Calibration curves for the determination of ethyl alcohol and acetaldehyde in human plasma.
| Analyte | Nominal concentration (µg/mL) | Peak area | Measured concentration (µg/mL) | Accuracy (%) | ||
|---|---|---|---|---|---|---|
| Analyte | tert-Butanol | Ratio | ||||
| Ethyl alcohol | Blank | 0 | 0 | - | - | - |
| 0 | 0 | 1,992,738 | - | - | - | |
| 20 | 296,242 | 1,989,242 | 0.149 | 20.2 | 100.8 | |
| 50 | 715,418 | 1,946,644 | 0.368 | 49.1 | 98.2 | |
| 100 | 1,448,401 | 1,928,653 | 0.751 | 99.9 | 99.9 | |
| 200 | 2,835,766 | 1,915,458 | 1.481 | 196 | 98.2 | |
| 500 | 7,368,672 | 1,926,049 | 3.826 | 507 | 101.4 | |
| 1,000 | 14,740,659 | 1,898,378 | 7.765 | 1,030 | 102.9 | |
| 2,000 | 28,945,048 | 1,944,926 | 14.88 | 1,970 | 98.6 | |
| y = 0.378x − 0.00334 | r2 = 0.9996 | |||||
| Acetaldehyde | Blank | 1,732 | 0 | - | - | - |
| 0 | 1,727 | 1,992,838 | - | - | - | |
| 0.2 | 13,490 | 1,989,242 | 0.007 | 0.200 | 100.0 | |
| 0.5 | 30,462 | 1,946,644 | 0.016 | 0.500 | 99.9 | |
| 1 | 58,669 | 1,928,653 | 0.030 | 0.999 | 99.8 | |
| 2 | 115,959 | 1,915,458 | 0.065 | 2.02 | 100.8 | |
| 5 | 288,942 | 1,926,049 | 0.150 | 5.04 | 100.8 | |
| 10 | 568,824 | 1,898,378 | 0.300 | 10.1 | 100.9 | |
| 20 | 1,127,561 | 1,944,926 | 0.580 | 19.6 | 97.8 | |
| y = 1.480x + 0.000863 | r2 = 0.9998 | |||||
Accuracy and precision
Method development for ethyl alcohol and acetaldehyde was subjected to intraday and interday validation of accuracy and precision. For ethyl alcohol, the intraday and interday accuracies ranged from 95.6% to 103.2% and 98.6% to 105.6%, respectively. The intraday and interday variations in precision ranged from 0.3% to 2.7% and 2.3% to 3.6%, respectively. For acetaldehyde, the intraday and interday accuracies ranged from 91.8% to 106.2% and 94.7% to 105.5%, respectively. The intraday and interday variations in precision ranged from 2.3% to 6.5% and 2.0% to 10.1%, respectively. The validation results of accuracy and precision are listed in Table 3.
Table 3. Accuracy and precision for the determination of ethyl alcohol and acetaldehyde in human plasma (n = 3).
| Analyte | Nominal concentration (µg/mL) | Intraday | Interday | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Mean of measured concentration (µg/mL) | SD | Accuracy (%) | Precision (%) | Mean of measured concentration (µg/mL) | SD | Accuracy (%) | Precision (%) | |||
| Ethyl alcohol | LLOQ | 20 | 20.60 | 0.5 | 103.2 | 2.4 | 20.80 | 0.5 | 104.0 | 2.3 |
| QC-Low | 60 | 57.40 | 0.8 | 95.6 | 1.5 | 59.20 | 1.8 | 98.6 | 3.0 | |
| QC-Medium | 400 | 391.0 | 11 | 97.8 | 2.7 | 398.0 | 12 | 99.6 | 3.0 | |
| QC-High | 1,600 | 1,616 | 5 | 101.0 | 0.3 | 1,689 | 60 | 105.6 | 3.6 | |
| Acetaldehyde | LLOQ | 0.2 | 0.212 | 0.005 | 106.2 | 2.3 | 0.211 | 0.004 | 105.5 | 2.0 |
| QC-Low | 0.6 | 0.606 | 0.013 | 98.2 | 2.4 | 0.613 | 0.028 | 102.2 | 4.5 | |
| QC-Medium | 4 | 3.700 | 0.12 | 92.6 | 3.2 | 3.790 | 0.27 | 94.7 | 7.0 | |
| QC-High | 16 | 14.70 | 1 | 91.8 | 6.5 | 15.30 | 1.5 | 95.5 | 10.1 | |
Accuracy (%) = (Mean of Measured Concentration/Nominal Concentration) × 100.
Precision (%) = (SD/Mean) × 100.
SD, standard deviation; LLOQ, lower limit of quantification; QC, quality control.
Stability
For the short-term stability of the samples (QC-Low and QC-High) left at room temperature for 12 hours, the following results were obtained: for ethyl alcohol, the accuracies were 95.3% and 101.9%, respectively, and the precision results were 1.9% and 0.6%, respectively; for acetaldehyde, the accuracies were 91.3% and 91.7%, respectively, and precision results were 1.2% and 0.4%, respectively. For the long-term stability of the samples (QC-Low and QC-High) stored in ultra-low temperature refrigeration for 7 days, the following results were obtained: for ethyl alcohol, the accuracies were 107.6% and 112.9%, respectively, and the precision results were 1.4% and 2.7%, respectively; for acetaldehyde, the accuracies were 103.9% and 101.9%, respectively, and the precision results were 0.7% and 1.6%, respectively. Finally, for the freeze and thaw stability of the samples that were stored in an ultra-low temperature refrigeration and then subjected to a freezing and thawing process twice (QC-Low and QC-High), the following results were obtained: for ethyl alcohol, the accuracies were 97.3% and 108.1%, respectively, and the precision results were 0.4% and 2.0%, respectively; for acetaldehyde, the accuracies were 98.6% and 95.6%, respectively, and the precision results were 1.8% and 1.7%, respectively. The validation results of sample stabilities are provided in Table 4.
Table 4. Stability for the determination of ethyl alcohol and acetaldehyde in human plasma (n = 3).
| Analyte | Nominal concentration (µg/mL) | Classification | Mean of measured concentration (µg/mL) | SD | Accuracy (%) | Precision (%) | |
|---|---|---|---|---|---|---|---|
| Ethyl alcohol | QC-Low | 60 | Short-term | 57.2 | 1.1 | 95.3 | 1.9 |
| Long-term | 64.6 | 0.9 | 107.6 | 1.4 | |||
| Freeze and thaw | 58.4 | 0.3 | 97.3 | 0.4 | |||
| QC-High | 1,600 | Short-term | 2,630 | 10 | 101.9 | 0.6 | |
| Long-term | 1,807 | 49 | 112.9 | 2.7 | |||
| Freeze and thaw | 1,730 | 35 | 108.1 | 2.0 | |||
| Acetaldehyde | QC-Low | 0.6 | Short-term | 0.548 | 0.007 | 91.3 | 1.2 |
| Long-term | 0.623 | 0.0005 | 103.9 | 0.7 | |||
| Freeze and thaw | 0.592 | 0.011 | 98.6 | 1.8 | |||
| QC-High | 16 | Short-term | 14.7 | 0.1 | 91.7 | 0.4 | |
| Long-term | 16.3 | 0.3 | 101.9 | 1.6 | |||
| Freeze and thaw | 15.3 | 0.3 | 95.6 | 1.7 | |||
Accuracy (%) = (Mean of Measured Concentration/Nominal Concentration) × 100.
Precision (%) = (SD/Mean) × 100.
SD, standard deviation; QC, quality control.
Carryover
The sample with the highest quantification limit concentration (StD7 in Table 1) was injected and run through the system; immediately afterward, the blank was injected and run through the system, and the peaks of ethyl alcohol, acetaldehyde and tert-butanol were not detected in the blank sample. Here, less than 20% of the peak area of the ethyl alcohol and acetaldehyde at the LLOQ (StD1 in Table 1) and less than 5.0% of the peak area of the tert-butanol were observed; thus, no carryover occurred in the sample analysis. The detailed results of carryover are listed in Table 5.
Table 5. Carryover for the determination of ethyl alcohol and acetaldehyde in human plasma (n = 3).
| Analyte | Injection time | Peak area of blank sample | Peak area of LLOQ sample | Peak area ratio (%) |
|---|---|---|---|---|
| Ethyl alcohol | 1st | 0 | 219,324 | 0.0 |
| 2nd | 0 | 0.0 | ||
| 3rd | 0 | 0.0 | ||
| Acetaldehyde | 1st | 1,344 | 10,856 | 12.4 |
| 2nd | 1,001 | 9.2 | ||
| 3rd | 0 | 0.0 | ||
| tert-Butanol | 1st | 0 | 1,440,510 | 0.0 |
| 2nd | 0 | 0.0 | ||
| 3rd | 0 | 0.0 |
Peak area ratio (%) = (Peak Area of Blank Sample/Peak Area of LLOQ Sample) × 100.
LLOQ, lower limit of quantification.
Batch size assessment
The accuracies of the average calculated concentrations for each QC concentration (60, 400 and 1,600 µg/mL in ethyl alcohol) of the continuously measured batches were 98.8% to 107.1%, 104.2% to 112.3% and 108.0% to 114.5%, respectively, and the precisions of the same batch samples were 0.9% to 3.4%, 0.4% to 3.7% and 0.2% to 3.2%, respectively. For acetaldehyde, the accuracies of the average calculated concentration for each QC concentration (0.6, 4 and 16 µg/mL in acetaldehyde) of continuously measured batch size assessment samples were 98.5% to 109.4%, 97.2% to 107.3% and 95.0% to 105.6%, respectively, and the precisions of the same batch sample were 0.7% to 4.0%, 1.0% to 5.4% and 0.7% to 5.2%, respectively. The validation results of the batch size assessment are listed in Table 6.
Table 6. Batch size assessment for the determination of ethyl alcohol and acetaldehyde in human plasma.
| Analyte | Nominal concentration (µg/mL) | Variable | Sample repeated injection cycles | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | ||||
| Ethyl alcohol | QC-Low | 60 | Mean | 59.3 | 60.5 | 60.6 | 62.4 | 61.3 | 64.3 | 62.3 | 63.0 | 61.2 |
| SD | 1.6 | 0.7 | 0.6 | 0.5 | 1.4 | 0.6 | 2.1 | 0.7 | 0.9 | |||
| Accuracy (%) | 98.8 | 100.9 | 101.1 | 104.0 | 102.2 | 107.1 | 103.8 | 104.9 | 102.0 | |||
| Precision (%) | 2.8 | 1.1 | 0.9 | 0.9 | 2.4 | 1.0 | 3.4 | 1.1 | 1.5 | |||
| QC-Medium | 400 | Mean | 417 | 422 | 428 | 426 | 449 | 433 | 444 | 434 | 434 | |
| SD | 2 | 10 | 4 | 12 | 17 | 2.9 | 1.1 | 0.7 | 1.6 | |||
| Accuracy (%) | 104.2 | 105.5 | 107.0 | 106.6 | 112.3 | 108.3 | 111.0 | 108.5 | 108.6 | |||
| Precision (%) | 0.4 | 2.5 | 1.0 | 2.8 | 3.7 | 2.9 | 1.1 | 0.7 | 1.6 | |||
| QC-High | 1,600 | Mean | 1,728 | 1,776 | 1,744 | 1,814 | 1,832 | 1,800 | 1,762 | 1,800 | 1,802 | |
| SD | 51 | 9 | 55 | 9 | 36 | 7 | 54 | 19 | 4 | |||
| Accuracy (%) | 108.0 | 111.0 | 109.0 | 113.4 | 114.5 | 112.5 | 110.1 | 112.5 | 112.6 | |||
| Precision (%) | 3.0 | 0.5 | 3.2 | 0.5 | 2.0 | 0.4 | 3.0 | 1.0 | 0.2 | |||
| Acetaldehyde | QC-Low | 0.6 | Mean | 0.630 | 0.625 | 0.656 | 0.640 | 0.591 | 0.632 | 0.643 | 0.606 | 0.646 |
| SD | 0.019 | 0.020 | 0.006 | 0.010 | 0.024 | 0.005 | 0.009 | 0.008 | 0.004 | |||
| Accuracy (%) | 104.9 | 104.2 | 109.4 | 106.6 | 98.5 | 105.3 | 107.2 | 101.0 | 107.7 | |||
| Precision (%) | 3.0 | 3.2 | 0.9 | 1.6 | 4.0 | 0.9 | 1.4 | 1.4 | 0.7 | |||
| QC-Medium | 4 | Mean | 4.07 | 3.89 | 4.15 | 3.98 | 4.20 | 4.29 | 4.24 | 3.93 | 4.17 | |
| SD | 0.10 | 0.10 | 0.04 | 0.10 | 0.23 | 0.11 | 0.07 | 0.05 | 0.08 | |||
| Accuracy (%) | 101.8 | 97.2 | 103.8 | 99.6 | 105.1 | 107.3 | 106.0 | 98.4 | 104.3 | |||
| Precision (%) | 2.5 | 2.7 | 1.0 | 2.6 | 5.4 | 2.6 | 1.7 | 1.3 | 2.0 | |||
| QC-High | 16 | Mean | 15.6 | 16.0 | 15.2 | 15.5 | 16.4 | 16.9 | 15.4 | 16.6 | 16.1 | |
| SD | 0.5 | 0.5 | 0.8 | 0.4 | 0.2 | 0.2 | 0.3 | 0.1 | 0.1 | |||
| Accuracy (%) | 97.6 | 99.8 | 95.0 | 96.6 | 102.3 | 105.6 | 96.5 | 104.0 | 100.9 | |||
| Precision (%) | 3.0 | 3.3 | 5.2 | 2.5 | 1.1 | 1.1 | 1.7 | 0.7 | 0.8 | |||
Number of total injection samples: 144 samples (including calibration curve samples).
Accuracy (%) = (Mean of Measured Concentration/Nominal Concentration) × 100.
Precision (%) = (SD/Mean) × 100.
SD, standard deviation; QC, quality control.
Pharmacokinetic study
Based on the pharmacokinetic analysis, when the placebo and HK-GCM-H01 were consumed, the blood concentration of alcohol reached the highest blood concentration at 1 hour and 1.5 hours, respectively, and the blood concentration of acetaldehyde reached the highest blood concentration at 0.5 hours after ingestion in both intake groups (Fig. 3). The half-life of acetaldehyde was 9.58 hours in the HK-GCM-H01 group and 13.16 hours in the placebo group. Although the HK-GCM-H01 group had a lower percentage than the placebo group, the results were not statistically significant. In addition, the Cmax values of ethyl alcohol of the HK-GCM-H01 and placebo groups were 964.74 ± 167.39 µg/mL and 980.30 ± 163.80 µg/mL, respectively, and the AUC0-15h values of ethyl alcohol were 3892.35 ± 745.35 h*µg/mL and 4003.59 ± 792.86 h*µg/mL, respectively. Finally, for baseline corrected acetaldehyde, the Cmax values of the HK-GCM-H01 and the placebo group were 0.56 ± 0.76 µg/mL and 0.61 ± 0.73 µg/mL, respectively, and the AUC0-15h values were 1.93 ± 2.57 h*µg/mL and 0.61 ± 0.73 h*µg/mL. Compared with those in the placebo group, the Cmax and AUC0-15h values of alcohol and aldehyde in all sample within the HK-GCM-H01 group were lower. The detailed results of the pharmacokinetic graph and parameters are presented in Fig. 3 and Table 7.
Figure 3. Mean plasma concentration-time curves for ethyl alcohol (A) and acetaldehyde (B) after oral administration of HK-GCM-H01 and placebo in human.
Table 7. Pharmacokinetic parameter of ethyl alcohol and acetaldehyde after oral administration of HK-GCM-H01 and placebo in human.
| Analyte | Classification | HK-GCM-H01 | Placebo | 95% CI | ||
|---|---|---|---|---|---|---|
| No. | Value | No. | Value | p-value* | ||
| Ethyl alcohol | Cmax (µg/mL) | 43 | 964.74 ± 167.39 | 43 | 980.30 ± 163.80 | 0.4323 |
| AUC0-15h (h·µg/mL) | 43 | 3,892.35 ± 745.35 | 43 | 4,003.59 ± 792.86 | 0.1137 | |
| Tmax (h)† | 43 | 1.00 (0.50–3.00) | 43 | 1.50 (0.50–3.00) | 0.8530 | |
| t1/2 (h) | 43 | 1.01 ± 0.38 | 43 | 1.07 ± 0.47 | 0.2273 | |
| Acetaldehyde | Cmax (µg/mL) | 43 | 0.64 ± 0.75 | 43 | 0.66 ± 0.72 | 0.8807 |
| AUC0-15h (h·µg/mL) | 37 | 3.95 ± 2.78 | 39 | 3.94 ± 2.76 | 0.5929 | |
| Tmax (h)† | 43 | 0.50 (0.00–3.00) | 43 | 0.50 (0.00–3.00) | 0.2592 | |
| t1/2 (h) | 33 | 9.58 ± 9.99 | 36 | 13.16 ± 17.22 | 0.3558 | |
| Baseline corrected acetaldehyde | Cmax (µg/mL) | 43 | 0.56 ± 0.76 | 43 | 0.61 ± 0.73 | 0.4878 |
| AUC0-15h (h·µg/mL) | 43 | 1.93 ± 2.57 | 43 | 2.22 ± 2.78 | 0.4003 | |
| Tmax (h)† | 43 | 0.50 (0.00–3.00) | 43 | 0.50 (0.00–3.00) | 0.2592 | |
| t1/2 (h) | 33 | 7.86 ± 11.39 | 36 | 11.53 ± 17.08 | 0.3696 | |
Values are presented as mean ± standard deviation or median (minimum–maximum).
CI, confidence interval; Cmax, maximum plasma concentration; AUC0-15h, area under the plasma concentration-time curve from 0 hour to 15 hours; Tmax, time to peak plasma concentration; t1/2, terminal half-life.
*Linear mixed effect model.
DISCUSSION
In this study, we aimed to develop an analytical method to quickly and simultaneously detect ethyl alcohol and its oxidized form, acetaldehyde, using HS-GC-MS/MS. Acetaldehyde in biosamples is volatile in the body; thus, headspace coupled GC-MS/MS was used to analyze acetaldehyde, and both ethyl alcohol and acetaldehyde could be clearly separated at 2.051 and 1.535 minutes, respectively (Fig. 1). In addition, headspace analysis was performed relatively simply on the plasma without a minimal preparation process. The tert-butanol was used as an IS and had a retention time of 2.721 minutes; as a result, the 3 analytes were quickly detected within a total run time of 4 minutes. The SIM mode was determined to detect ethyl alcohol (m/z 45.0 → m/z 31.0), acetaldehyde (m/z 43.0 → m/z 29.0) and tert-butanol (m/z 59.0 → m/z 41.0) with better sensitivity, and the mass to charge for detecting each analyte was quantified using m/z 31.0, 29.0 and 41.0, respectively (Fig. 1). After setting up the sample pretreatment method and analysis conditions, we performed method validation and reviewed whether the system was suitable for the analysis of ethyl alcohol and acetaldehyde. As a pre-validation process, the LLOQ samples were injected once a day for a total of 3 days to confirm the precision of the peak area ratio of each analyte (ethyl alcohol and acetaldehyde) and IS (tert-butanol) and the retention time. In terms of system suitability, the precision of the peak area ratio for ethyl alcohol and tert-butanol ranged from 0.4% to 2.9%, and the precision results from the retention times were 0.0% and 0.0% for ethyl alcohol and tert-butanol, respectively. In addition, the precision of the peak area ratio for acetaldehyde and tert-butanol ranged from 0.1% to 2.2%, and the precision results from the retention times were 0.0% and 0.0% for acetaldehyde and tert-butanol, respectively. Because the precision confirmed for all analytes was less than 10%, validation for the development of this analysis method was possible (data not shown).
The analytical method developed in this study was validated through evaluation of linearity, accuracy and precision, stability and carryover, and batch size. All validation results revealed that the accuracy was within 100.0 ± 15.0%, and the precision was 15.0% or less; additionally, for the lowest quantification limit, the accuracy was within 100.0 ± 20.0%, and the precision was 20.0% or less. All validation results obtained in this study meet the acceptance criteria presented in ‘FDA guidance for industry: bioanalytical method validation’ [21] and ‘KFDA guidance for industry: bioanalytical method validation’ [22].
The application of developed analytical method was intended to determine the pharmacokinetic properties from the placebo and HK-GCM-H01 administered in healthy adult volunteers. The trial was conducted in the form of a randomized, double-blind, placebo-controlled, single-dose, crossover design trial, and the plasma of 43 out of 50 subjects was analyzed. As a result of the analysis, the blood concentration of alcohol reached the highest blood concentration was 1 hour and 1.5 hours, respectively after ingestion of placebo and HK-GCM-H01, but there was no significant difference (p = 0.8530) (Table 7). The baseline corrected acetaldehyde of half-life was 7.86 hours in the HK-GCM-H01 group and 11.53 hours in the placebo group. Although lower results were observed in the HK-GCM-H01 group compared to the placebo group, there was no statistically significant difference between the ingestion groups (p = 0.3696). As is well known, acetaldehyde is a biomarker that can evaluate the degree of hangover [5], and the degree of hangover of HK-GCM-H01 was evaluated through this PK study applied to human plasma. As proven in the above study, the analytical method of ethyl alcohol and acetaldehyde developed in this study can be used as the fastest and simplest method to evaluate the degree of hangover relief in the blood.
In this experiment, an analysis method for ethyl alcohol and acetaldehyde in human plasma was developed using HS-GC-MS/MS, and validation was performed. Based on the evaluation of the linearity, accuracy and precision, carry-over, and batch size for validation of the analytical method, all of these factors satisfied the acceptance criteria according to FDA guidelines, and the method was determined to be valid. In addition, the stability of ethyl alcohol and acetaldehyde in plasma did not have a significant effect on the quantitative results when the samples were stored at room temperature for less than 12 hours, stored in cryogenic freezers (−85°C to −65°C) for less than 7 days, or frozen and thawed two times during cryogenic freezer storage. As a result of the batch size assessment, a total of 144 samples for the ethyl alcohol and acetaldehyde determination are able to measured, including standard samples for the blank, zero and calibration curves. Finally, using the results of the above analytical method, we performed a pharmacokinetic study on human plasma and applied ethyl alcohol and acetaldehyde to the analysis. No statistically significant difference in alcohol or acetaldehyde content was observed between the placebo and HK-GCM-H01 groups. Our development method could be used for regular ethyl alcohol and acetaldehyde analysis to determine the clinical effect of alcohol metabolism in biological samples as well as plasma; thus, this method has potential applications in diverse fields, such as medications and forensics.
Footnotes
Funding: This study was sponsored by HK inno.N Corp., Seoul, Republic of Korea.
Conflict of Interest: - Authors: Nothing to declare
- Reviewers: Nothing to declare
- Editors: Nothing to declare
Reviewer: This article was reviewed by peer experts who are not TCP editors.
- Conceptualization: Oh HA, Park MK.
- Data curation: Oh HA.
- Formal analysis: Oh HA, Park MK.
- Methodology: Oh HA.
- Investigation: Park MK.
- Project administration: Oh HA, Park MK.
- Resources: Oh HA, Park MK.
- Software: Oh HA.
- Supervision: Park MK.
- Validation: Oh HA, Park MK.
- Visualization: Oh HA.
- Writing - original draft: Oh HA.
SUPPLEMENTARY MATERIAL
Flow chart of the clinical study
References
- 1.Simpson G. Medicolegal alcohol determination: Widmark revisited. Clin Chem. 1988;34:888–889. [PubMed] [Google Scholar]
- 2.Zekan P, Ljubičić N, Blagaić V, Dolanc I, Jonjić A, Čoklo M, et al. Pharmacokinetic analysis of ethanol in a human study: new modification of mathematic model. Toxics. 2023;11:793. doi: 10.3390/toxics11090793. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Bauer R, Cowan DA, Crouch A. Acrolein in wine: importance of 3-hydroxypropionaldehyde and derivatives in production and detection. J Agric Food Chem. 2010;58:3243–3250. doi: 10.1021/jf9041112. [DOI] [PubMed] [Google Scholar]
- 4.Yokoyama A, Tsutsumi E, Imazeki H, Suwa Y, Nakamura C, Mizukami T, et al. Salivary acetaldehyde concentration according to alcoholic beverage consumed and aldehyde dehydrogenase-2 genotype. Alcohol Clin Exp Res. 2008;32:1607–1614. doi: 10.1111/j.1530-0277.2008.00739.x. [DOI] [PubMed] [Google Scholar]
- 5.Zakhari S. Overview: how is alcohol metabolized by the body? Alcohol Res Health. 2006;29:245–254. [PMC free article] [PubMed] [Google Scholar]
- 6.Edenberg HJ. The genetics of alcohol metabolism: role of alcohol dehydrogenase and aldehyde dehydrogenase variants. Alcohol Res Health. 2007;30:5–13. [PMC free article] [PubMed] [Google Scholar]
- 7.Uchida K. Role of reactive aldehyde in cardiovascular diseases. Free Radic Biol Med. 2000;28:1685–1696. doi: 10.1016/s0891-5849(00)00226-4. [DOI] [PubMed] [Google Scholar]
- 8.Barrera G, Pizzimenti S, Daga M, Dianzani C, Arcaro A, Cetrangolo GP, et al. Lipid peroxidation-derived aldehydes, 4-hydroxynonenal and malondialdehyde in aging-related disorders. Antioxidants (Basel) 2018;7:102. doi: 10.3390/antiox7080102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Cairns HS, Rideout JM, Peters TJ, Laker MF, Mansell MA. Changes in blood acetaldehyde concentrations during acetate haemodialysis. Nephrol Dial Transplant. 1988;3:637–640. doi: 10.1093/oxfordjournals.ndt.a091719. [DOI] [PubMed] [Google Scholar]
- 10.Su J, Wang P, Zhou W, Peydayesh M, Zhou J, Jin T, et al. Single-site iron-anchored amyloid hydrogels as catalytic platforms for alcohol detoxification. Nat Nanotechnol. 2024;19:1168–1177. doi: 10.1038/s41565-024-01657-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Tiscione NB, Alford I, Yeatman DT, Shan X. Ethanol analysis by headspace gas chromatography with simultaneous flame-ionization and mass spectrometry detection. J Anal Toxicol. 2011;35:501–511. doi: 10.1093/anatox/35.7.501. [DOI] [PubMed] [Google Scholar]
- 12.Açikgöz G, Hamamci B, Yildiz A. Determination of ethanol in blood samples using partial least square regression applied to surface enhanced raman spectroscopy. Toxicol Res. 2018;34:127–132. doi: 10.5487/TR.2018.34.2.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Wunder C, Pogoda W, Paulke A, Toennes SW. Assay of ethanol and congener alcohols in serum and beverages by headspace gas chromatography/mass spectrometry. MethodsX. 2021;8:101563. doi: 10.1016/j.mex.2021.101563. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Heit C, Eriksson P, Thompson DC, Charkoftaki G, Fritz KS, Vasiliou V. Quantification of neural ethanol and acetaldehyde using headspace GC-MS. Alcohol Clin Exp Res. 2016;40:1825–1831. doi: 10.1111/acer.13156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.McCarver-May DG, Durisin L. An accurate, automated, simultaneous gas chromatographic headspace measurement of whole blood ethanol and acetaldehyde for human in vivo studies. J Anal Toxicol. 1997;21:134–141. doi: 10.1093/jat/21.2.134. [DOI] [PubMed] [Google Scholar]
- 16.Pontes H, Guedes de Pinho P, Casal S, Carmo H, Santos A, Magalhães T, et al. GC determination of acetone, acetaldehyde, ethanol, and methanol in biological matrices and cell culture. J Chromatogr Sci. 2009;47:272–278. doi: 10.1093/chromsci/47.4.272. [DOI] [PubMed] [Google Scholar]
- 17.Brien JF, Loomis CW. Gas-liquid chromatographic determination of ethanol and acetaldehyde in blood. Clin Chim Acta. 1978;87:175–180. doi: 10.1016/0009-8981(78)90073-6. [DOI] [PubMed] [Google Scholar]
- 18.Dator RP, Solivio MJ, Villalta PW, Balbo S. Bioanalytical and mass spectrometric methods for aldehyde profiling in biological fluids. Toxics. 2019;7:32. doi: 10.3390/toxics7020032. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Cordell RL, Pandya H, Hubbard M, Turner MA, Monks PS. GC-MS analysis of ethanol and other volatile compounds in micro-volume blood samples--quantifying neonatal exposure. Anal Bioanal Chem. 2013;405:4139–4147. doi: 10.1007/s00216-013-6809-1. [DOI] [PubMed] [Google Scholar]
- 20.Murakami T, Iwamuro Y, Sakamoto Y, Minami E, Ishimaru R, Tsuchihashi H, et al. Rapid simultaneous determination of cyanide, azide, and ethanol in whole blood using headspace gas chromatography-mass spectrometry. Chromatographia. 2023;86:701–706. [Google Scholar]
- 21.U.S. Food and Drug Administration. Bioanalytical method validation guidance for industry [Internet] [Accessed September 19, 2024]. https://www.fda.gov/media/70858/download .
- 22.Ministry of Food and Drug Safety (KR) Biological sample analysis method validation and test specimen analysis guidelines [Internet] [Accessed September 19, 2024]. https://www.mfds.go.kr/brd/m_1060/view.do?seq=13054&srchFr=&srchTo=&srchWord=&srchTp=&itm_seq_1=0&itm_seq_2=0&multi_itm_seq=0&company_cd=&company_nm=&page=1 .
Associated Data
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Supplementary Materials
Flow chart of the clinical study