Abstract
Background
Determination of ethanol levels in aircraft accident victims constitutes an important part of investigation. However, postmortem production of alcohol by microbial fermentation is known to interfere with the results. Distinguishing postmortem produced alcohols from antemortem ingested is very important in interpretation of results. Ratio of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA) metabolites of serotonin, has known to provide a convenient, rapid, and reliable solution as antemortem ethanol leads to an elevation in the 5-HTOL/5-HIAA ratio after ingestion of alcohol (5-HTOL/5-HIAA = >15 pm/nm).
Methods
Triple quadruple (QQQ) liquid chromatography–mass spectrometry (LC-MS) with electrospray ionization positive mode has been used for development of single tube multiple reaction monitoring (MRM) method for simultaneous quantification of 5-HTOL and 5-HIAA in urine. Deglucuronidation of 5-HTOL glucuronide in urine by beta-glucuronidase followed by simple sample preparation has been adopted. Examination of the ratio on urine samples from 15 individuals after consumption of 60 and 90 ml of whiskey has been carried out at different time interval.
Results
A single method for analysis of both the analytes was developed with sensitivity of 50 ppb and recovery of around 80–90%. Examination of the ratio on urine samples revealed that the ratio was >15 in all groups consuming 60 ml and 90-ml whiskey up to 12 h after alcohol ingestion.
Conclusion
This is a unique highly sensitive single LC-MS method, which has been developed for simultaneous estimation of both 5-HTOL and 5-HIAA on same instrument for proving antemortem alcohol ingestion with high degree of sensitivity and specificity.
Keywords: Air crash investigation, Ethanol, Serotonin metabolites, Biomarkers
Introduction
Toxicological analysis of air crash victims includes analysis of ethanol levels in blood. Several studies1, 2, 3, 4, 5 have indicated alcohol as a major cause of human error, leading to aircraft accidents. The concentration blood alcohol in these studies ranges from trace amounts to 420 mg/dL.6
Identification of alcohol/ethanol in blood of air crash victims lead to other issues such as legal and administrative actions as per the concentration of alcohol. Widely used method for analysis of ethanol is by gas chromatography with head space sampler (GC-HSS). GC-HSS is the commonly used technique in postmortem specimens of all air crash victims. However, microbial fermentation postmortem could also increase the level of alcohol in the samples being tested, leading to erroneous interpretation of the results.
This is the most important confounding factor during interpretation of such ethanol-positive results. The ethanol produced owing to microbial fermentation in postmortem specimens is the common confounding factor encountered during reporting of such results. Many studies have highlighted the importance of alcohol production after death.7 In several instances, high level of ethanol have been found in postmortem specimens’ without any antemortem ingestion of alcohol. Therefore simple postmortem ethanol levels cannot be used as exclusive proof of antemortem ingestion. Many species of bacteria, yeast, and molds are capable of producing postmortem ethanol of which Candida albicans and Eschericha coli are prominent.7 Even though the production of alcohol postmortem by organisms can be reduced by maintaining a cold chain or using NaF, these methods do not completely eliminate the production of alcohol postmortem8,9.
5-Hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA) are metabolites of serotonin (5-HT) and are known to be the best biomarkers for antemortem ethanol ingestion and have generated lot of interest in the field of forensic toxicology.10
The metabolism of 5-HT initially involves oxidative deamination to form the intermediate aldehyde, 5-hydroxyindole-3-acetaldehyde (5-HIAL). The 5-HIAL is oxidized by aldehyde dehydrogenase (ALDH) to form 5-HIAA, a major metabolite of 5-HT. 5-HIAL can also be reduced by alcohol dehydrogenase (ADH) and catalyzed by aldehyde reductase to form 5-HTOL, a relatively minor metabolite of 5-HT (Fig. 1). However, ethanol consumption has been shown to lead to a significantly enhanced production of 5-HTOL and to reduce the formation of 5-HIAA as the enzyme ALDH and ADH are common to both alcohol metabolism and formation of 5-HIAA and 5-HTOL from 5-HT. Ethanol metabolism increases the NADH/NAD + ratio and increasing the production of 5-HTOL and decreasing 5-HIAA, thus increasing the ratio. An elevated ratio of 5-HTOL/5-HIAA (>15 pm/nm) in urine is a clear indication of acute ingestion of alcohol before death.11,12
Fig. 1.
The metabolism of serotonin.
The liquid chromatography–mass spectrometry (LC-MS) is a diagnostic analytical tool for analysis of many compounds and depends on the type of MS used in the equipment. The triple quadruple MS is used for detection and quantification of small compounds by adopting MRM mode. The MS analyze the mass of the compounds after ionization. The mass filter assesses the molecular mass of parent compound and its product ions for identification and quantification is carried out by calculating the area under the curve.
This study was undertaken with an objective of developing a sensitive and specific LC-MS method for detection and quantification of 5-HTOL and 5-HIAA in urine and to evaluate the ratio of 5-HTOL/5-HIAA in cases of alcohol ingestion, as a marker for recent alcohol intake.
Materials and methods
Agilent 1260 infinity HPLC (Agilent Technologies) along with Agilent QQQ Mass spectrometry was used for developing LC-MS protocols for biomarkers. The Vac Elut 12 Manifold from Agilent (P/N: 5982–9110) was used for solid-phase extraction (SPE) during sample preparation and the nitrogen gas–based sample concentrator was used to concentrate the elutes of the SPE before injection to system.
The pure compounds/standards for method development used were 5-HTOL molecular weight, 177.2 g/mol (sc-217202) from Santa Cruz Biotechnology (Dallas, Texas, USA) and 5-HIAA molecular weight, 191.2 g/mol (H8876-1G) from Sigma Life Sciences. All solvents including acetonitrile, methanol, and formic acid used in the present study were of LC-MS grade from Sigma-Aldrich, USA.
Urine samples used to validate/test the developed protocols on actual biological specimen were obtained from 15 healthy individuals. The urine samples were collected at different time interval (6hr, 12hr, 18hr, 24hr, 48hr, and 72hr) after consumption of 60 ml and 90 ml of whiskey. Urine samples collected from healthy subjects without previous intake of alcohol were used as negative controls.
The pure compounds were weighed and reconstituted in LC-MS grade methanol in which the particular compounds are freely soluble. From a stock concentration of 1 mg/ml, dilutions were used for developing multiple reaction monitoring (MRM) transitions and method development on column. Individually, each compound at known minimum concentration was infused directly to mass spectrometer along with mobile phase for developing MRM transitions. For both the compounds, the electrospray ionization (ESI) was used and tried both positive and negative mode of ionization. Different fragmentor voltages, different collision energies for production of product ions were evaluated as a part of protocol development to create MRM transitions unique to each compound (Table 1). These MRM transitions on MS were tabulated for both the compounds.
Table 1.
Details of MRM transitions developed for the 5-HTOL and 5-HIAA used for method development on column.
| Compound | Precursor ion | Product ion(s) | Fragmentor voltage (V) | Collision energy (eV) | Polarity |
|---|---|---|---|---|---|
| 5-HTOL | 178.3 | 160.2, 132.2 | 100 | 8, 15 | Positive |
| 5-HIAA | 192.4 | 146.2 | 100 | 10 | Positive |
5-HTOL, 5-hydroxytryptophol; 5-HIAA, 5-hydroxyindole-3-acetic acid.
5-HTOL and 5-HIAA: LC-MS conditions
Initially the chromatographic separation and method development was attempted separately for 5-HTOL and 5-HIAA compounds. After developing separate methods for both the metabolites, a single method (Mix MRM) for both the analytes on a same column was attempted using an Agilent 1260 infinity HPLC equipped with a Zorbax Eclipse plus C18 (3.0 × 100 mm, 1.8 μm P/N: 959964–30) analytical column. Samples were injected using an Agilent G1367C autosampler. Identification and quantification of the compounds were accomplished using Agilent 6430 triple quadrupole mass spectrometer. For all determinations, the HPLC was operated in a gradient mode with a constant flow rate of 0.30 ml/min. The mobile phase used consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient was set up as follows: initial mobile phase composition of 75% A and 25% B which changed to 5% of A and 95% of B in 3 min. The same composition was held up to 4.1 min. The mobile phase composition changed to initial composition from 4.1 to 5 min. The postrun time was kept as 2 min.
The sample injection volume was held constant at 1 μL. The operating conditions for the data collection segments of the MS were as follows:
Mode: ESI positive mode, gas temp: 300 °C, gas flow:10 l/min, nebulizer: 35psi, capillary: 3500V, Delta EMV: 400V and cycle time: 500 ms.
Control of the HPLC system, integration of any chromatographic peaks, and communication with the mass spectrometer were accomplished via a personal computer using QQQ data acquisition software (Agilent Technologies Pvt ltd).
Preparation of matrix-matched standards and calibration: 5-HTOL and 5-HIAA Mix MRM method
A series of calibration standards were prepared in saline solution by spiking 50, 100, 200, 400, and 800 ppb of aqueous 5-HTOL and 5-HIAA mix in a microfuge tube. Saline spiked with acetonitrile was used as blank. In brief, 750 μL of acetonitrile was added to 100 μL of saline spiked with known concentration of standard mix. The mix was vertexed for 30 s and centrifuged at 6000 rpm for 5 min at 8°C. The supernatant was transferred to Bond Elut Captiva ND Lipid cartridges. The vacuum was applied and the elute was collected. The elute was subjected to vacuum concentration using N2 concentrator and finally reconstituted with 200 μL of acetonitrile + water (1:1) with 0.1% formic acid before injecting into the system.
Sample preparation method standardized for urine samples
Urine sample from participants who had consumed alcohol were hydrolyzed with 50ul of β-glucuronidase enzyme (12,500 units) before processing through Captiva ND lipid cartridges. In brief, 100 μL of urine along with 50 μL of enzyme and 100 μL of sodium acetate buffer was incubated at 37 °C for 1 h before processing through Bond Elut Captiva ND Lipid cartridges as explained previously. The elute was concentrated using N2 concentrator and reconstituted with 200 μL of acetonitrile + water (1:1) with 0.1% formic acid and injected into the system.
Validation of method was carried out on 15 urine samples of alcohol-ingested persons by determining the 5-HTOL/5-HIAA ratio.
Results
The MRM transitions for 5-HTOL and 5-HIAA using ESI-positive mode was developed before developing chromatographic methods (Table 1) which are unique and specific to each compound. As mentioned in the methods, initially separate methods were developed for both 5-HTOL and 5-HIAA on separate column with different chromatographic conditions. However, the results of the separated methods were not included in the manuscript.
Mix MRM method for simultaneous detection of both 5-HTOL and 5-HIAA
After developing separate methods for both analytes, a single method for analysis of both the analytes on same column was successfully developed and demonstrated for simultaneous detection and quantification of 5-HTOL and 5-HIAA from urine sample.
Chromatographic separation was optimized for both 5-HTOL and 5-HIAA on the same column with the same retension time (Rt) for both the compounds. MassHunter Optimizer was used to identify the most abundant MRM transitions for both the target compounds. Both the compounds showed good signal responses in the ESI-positive ion mode. A simple sample preparation method was developed in the present study to extract both the compounds from urine samples after enzymatic hydrolysis of urine revealed a recovery of 80–90%. The run time of less than 5 min was standardized (Fig. 2) for determination of both the compounds.
Fig. 2.
Total ion chromatogram of 200 ng/mL calibration standard mix of 5-HTOL and 5-HIAA compounds. 5-HTOL, 5-hydroxytryptophol; 5-hydroxyindole-3-acetic acid.
Matrix-matched calibration curves were generated with linear curve fitting and were weighed (1/x). A linear dynamic range of 50–800 ng/mL was achieved for both the compounds with an R2 value greater than 0.9. Fig. 3 depicts calibration curves of both compounds.
Fig. 3.
Matrix-matched calibration curves of 5-HTOL and 5-HIAA. 5-HTOL, 5-hydroxytryptophol; 5-hydroxyindole-3-acetic acid.
The method validation carried out by calibration revealed a working range of 50–800 ppb. The CV was >0.995 with sensitivity of 50 ppb for both the compounds. The repeatability study revealed similar retention time, peak shape, and response after 6 injections. The specificity of the developed method was attributed to the mass of precursor ions, Rt, product ions, and ion ratio.
The ratio of 5-HTOL/5-HIAA was calculated on urine samples collected at different time interval (6 h, 12 h, 18 h, 24 h, 48 h, and 72 h). We found that the ratio of 5-HTOL/5-HIAA was >15 upto 12 h after alcohol consumption in both groups ingesting 60 ml and 90 ml whiskey-ingested individuals.
The mean ± SD value of the 5-HTOL/5-HIAA ratio (pm/nm)at 6hrs and 12 h after ingestion of 60 and 90 ml whisky was elevated well above 15 and was nil for negative controls (Table 2). The ratio of 5-HTOL/5-HIAA was <15 after 12 h of consumption of alcohol both in 60 ml and 90 ml ingested individuals (Fig. 4).
Table 2.
Mean 5-HTOL/5-HIAA ratio (pm/nm) at various interval of time.
| Hours | After 60 ml of whiskey |
After 90 ml of whiskey |
||
|---|---|---|---|---|
| Mean | SD | Mean | SD | |
| 6 | 40 | 10.7 | 104 | 23.1 |
| 12 | 22.9 | 5.4 | 51.4 | 16.3 |
| 18 | Nil | – | 2.71 | 0.8 |
| 24 | Nil | – | Nil | – |
| 48 | Nil | – | Nil | – |
| 72 | Nil | – | Nil | – |
5-HTOL, 5- hydroxytryptophol; 5-hydroxyindole-3-acetic acid; SD, standard deviation.
Fig. 4.
The mean ratio of 5-HTOL/5-HIAA at different time interval after consumption of 60 ml and 90 ml of whiskey. 5-HTOL, 5-hydroxytryptophol; 5-hydroxyindole-3-acetic acid.
Discussion
The present study addresses the issue of ethanol production after death in the victims of fatal aircraft accidents, which leads to false positive results and creates problems in interpretation of results. The accurate estimation of the ratio of metabolites of 5-HT in urine, by QQQ LC-MS with ESI-positive mode will establish consumption and metabolism of alcohol by the body beyond doubt. 5-HTOL and 5-HIAA are the metabolites of 5-HT and ratio of 5-HTOL/5-HIAA> 15 pm/nm in urine conclusively establishes recent alcohol ingestion.11 Therefore, the 5-HTOL/5-HIAA ratio has been investigated as a marker for recent alcohol ingestion instead of individual alone. This established procedure when used in the actual scenario of aircraft accident, where there are high chances of microbial formation of ethanol after death, can adequately differentiate ethanol produced from microbial fermentation from ethanol ingested by the pilot before crash. This is therefore a very useful tool in aircraft accident investigation.10
There are no established enzymatic or ELISA platforms to estimate the ratio of 5-HT metabolites. Chromatographic separation followed by detection is a better approach to analyze these metabolites. Unlike drugs, estimation of these metabolites is complicated. Currently, the levels of 5-HTOL and 5-HIAA in individual samples have been estimated using two completely different analytical tools – gas chromatography–mass spectrometry (GC-MS) for 5-HTOL and LC-MS for 5-HIAA. Concentrations of the major metabolite 5-HIAA was measured using liquid chromatography with isocratic elution and electrochemical detection (LC–EC).13 However, the minor metabolite 5-HTOL generally gets excreted as 5-HTOL glucuronide (GTOL), in a glucuronidated form, in the urine. Therefore, the detection of 5-HTOL involves two approaches. The first method was to detect the GTOL compound directly in urine, but this has problems of unavailability of pure standards of GTOL and cost of chemical synthesis of GTOL for method development. The second method is deglucuronidation or hydrolysis of GTOL to 5-HTOL and estimation of 5-HTOL by GC-MS detection after derivatization,14 as methods using LC–EC could not measure the very small quantities of this substance accurately. Consequently, the use of two different analytical techniques to get the 5-HTOL/5-HIAA ratio in a specimen decreases the precision and there by reliability of the final result.
There has been one study on a single method/approach for detection of both these compounds in urine. Johnson et al. at Federal Aviation Administration, Civil Aerospace Medical Institute, USA, developed a single analytical approach for simultaneous detection of 5-HTOL and 5-HIAA using an HPLC equipped with C-8 guard column and atmospheric pressure chemical ionization (APCI) coupled with ion trap mass spectrometer. In their method for all determinations, HPLC was operated on isocratic mode and 5-HTOL and 5-HIAA were derivatized with trimethylsilyl (TMS) before detection for better sensitivity. The study also revealed that antemortem ingestion of ethanol leads to an elevation in the 5-HTOL/5-HIAA ratio up to 11–19 h after acute ingestion.14 In the present study, we describe a remarkably a precise method with very low detection limits for the rapid and simultaneous determination of 5-HTOL and 5-HIAA on the same equipment ie. single LC-MS detection. The present study adopted a simple hydrolysis procedure for deglucuronidation of GTOL to 5-HTOL before analyzing the 5-HTOL without derivatization of 5-HTOL but still achieved the required sensitivity. Further, the present study is the first of its kind for simultaneous analysis of both 5-HTOL and 5-HIAA in urine on single platform.
In the present study, Agilent 6430 Triple Quadrupole LC-MS with ESI-positive mode was used for determination of 5-HTOL and 5-HIAA in urine.
Single method for simultaneous detection and quantification of both analytes has been developed with sensitivity of 50 ppb and recovery of around 85%. Chromatographic separation was optimized for both 5-HTOL and 5-HIAA on the same column with same Rt as the specificity is based on the molecular mass, product ions, and the ratio between the ions in MRM method. Therefore, it is always possible to develop a single method for multiple analytes having same retention time (ie same peak) but having different mass. This is the advantage of MS with conventional detectors when analyzing multiple compounds with same Rt. It was important and very much essential to develop a single method for analyzing both the metabolites on same instrument and on same column to exclude different confounding factors and to make the estimation more accurate and reliable as the ratio of both the compounds in urine will be ultimately used for determination of antemortem ingestion. Analysis of both compounds in different equipment/method/column may affect the final ratio. Bond Elut Captiva ND Lipid Cartridges were used for sample clean-up process after enzymatic hydrolysis of urine sample for deglucurination. Saline was used for matrix-matched calibration instead of blank urine as normal healthy person urine may also contain trace amounts of 5-HIAA affecting the calibration standards. The range of calibration used was 50–800 ppb for both compounds. Examination of the 5-HTOL/5-HIAA ratio on 15 urine samples of alcohol-ingested individual revealed a ratio >15 upto 12 h after ingestion of alcohol, which is in concurrence with the previous published literature.11
Our protocol and the only other single method developed for both these analytes by the Federal Aviation Administration (FAA) shows many significant differences. We used ESI technique for ionization and triple quadruple MS for detection and quantification of both the analytes compared with of APCI ionization and ion trap MS used by the FAA study. The separation in the present study was operated in gradient mode as compared with isocratic mode. The method developed by us was much more simpler as it had a simple sample preparation technique without derivatizing the analytes before analysis, as compared with the laborious TMS derivatization procedure adopted by the FAA study. The FAA study reported the elevation in the 5-HTOL/5-HIAA ratio for 11–19 h after acute alcohol ingestion which is in concurrence with the present study.11 All the aforementioned reasons make this method a unique, simple, and novel one.
Conclusion
A simple distinctive and original method for determination of the ratio of 5-HTOL and 5-HIAA in urine was developed and validated as tool for confirming recent alcohol intake. The method is unique as both the analytes can be estimated by a single method and on the same instrument, excluding chances of error and bias. The study showed an elevated ratio 5-HTOL/5-HIAA upto 12 h after ingestion of alcohol. Now this ratio can be used in actual aircraft accident investigation situations on samples from victims with high ethanol levels. This method, being based on LC-MS, has a very high degree of sensitivity and can conclusively prove or disprove the source of the ethanol present in the sample. This study is distinctive in developing a single simultaneous method on for both analytes on LC-MS which is much simpler, yet retaining the required sensitivity as compared with the other methods. In this study, the only of its kind in India, both 5-HTOL and 5-HIAA are detected without derivatization of parent compounds. This ratio, being a highly sensitive and specific biomarker of recent alcohol intake, one can prove or disprove antemortem alcohol consumption and help confidently analyze all ethanol-positive victims of air crash, interpret the high values, and decrease the false positives resulting from postmortem production.
Conflicts of interest
The authors have none to declare.
Acknowledgment
This paper is based on Armed Forces Medical Research Committee Project No. 4538/2014 granted and funded by the office of the Directorate General Armed Forces Medical Services and Defence Research Development Organisation, Government of India.
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