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Journal of Chromatography B 1100–1101 (2018) 33–38
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Novel zwitterionic HILIC stationary phase for the determination of ethyl glucuronide in human hair by LC-MS/MS
T
Diego Palumboa, Paolo Faisb, Anna Calìc, Monica Lusardic, Elisabetta Bertola, ⁎ Jennifer P. Pascalia, a
Forensic Toxicology Division, Department of Health Sciences, University of Florence, Largo Brambilla 3, 50134 Florence, Italy University of Bologna, Department of Medical and Surgical Sciences, Unit of Legal Medicine, Via Irnerio 49, Bologna, Italy c Agilent Technologies, Via V. Lamaro, Rome, Italy b
ARTICLE INFO
ABSTRACT
Keywords: Ethyl glucuronide Liquid chromatography Hair Hydrophilic chromatography Alcohol HILIC-Z
Some recent studies have described a shift from traditional reversed-phase to more hydrophilic LC chemistry for EtG determination in hair (hEtG). The reason relies on the poor retention of C8– and C18-based columns for polar compounds, even in presence of great amount of aqueous phase. This work presents the development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-LC-MS/MS) method based on a novel zwitterionic stationary phase for the analysis of hEtG. The linearity was assessed in the range of 5–100 pg/mg hair, with a correlation coefficient of > 0.99. The method was selective and sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1.4 pg/mg and 4.5 pg/mg in hair, respectively. Suitable diagnostic sensitivity was achieved without the introduction of a sample purification step, or a post column solvent addition. The method was successfully applied to real hair samples after full validation. This method, based on a separation at neutral conditions, confirmed the optimum retention and thus selectivity for weak acids in zwitterionic HILIC columns.
1. Introduction Alcohol is the most widely consumed psychoactive substance and is becoming a problematic addiction issue in millions of people worldwide. In fact, unhealthy alcohol use can be either a primary or a secondary cause of liver disease and besides medical complications its abuse can be responsible of severe social problems [1,2]. Additionally, from the forensic point of view, it is of extreme importance to monitor alcohol abstinence in patients undergoing an alcohol withdrawal treatment when a legal cause is undergoing. Laboratory testing traditionally based diagnosis on the assessment of biomarkers such as mean corpuscular volume (MCV), carbohydrate deficient transferrin (CDT) and liver enzymes (gamma glutamyl-transferase, aspartate aminotransferase and alanine aminotransferase, aspartate-amine-transferase and alanine-amine-transferase). In the last few years, increased attention has been paid to ethyl glucuronide (EtG), a new specific alcohol intake marker [3]. It is a direct metabolite of non-oxidative breakdown of ethanol accounting for < 0.1% of total ethanol elimination. Through the measurement of EtG in hair (hEtG), it is possible to assess both the chronic alcohol abuse over time and to document the treatment efficacy of patients in a withdrawal program. However, due to its high polarity, ⁎
EtG is incorporated into hair only in very small amounts and thus the reliable detection of EtG in hair requires sensitive techniques in reason of the low concentrations (pg/mg range) finally present in the matrix, even in presence of abuse behaviours. Previous researches have shown that analytical methods based on gas chromatography (GC) with mass spectrometry (MS) and liquid chromatography (LC) with MS are the techniques of choice and, to reach appropriate sensitivity, sample preparation based on solid-phase extraction (SPE) is, in most cases, applied to obtain cleaner extracts. The recommended cut-off levels for hEtG diagnostic purposes, suggested by the Society of Hair Testing (SoHT), are substantially two values: 30 pg/mg to distinguish from moderate and heavy alcohol consumption and below 7 pg/mg to pass the medical assessment of alcohol abstinence [4]. A small overview on analytical methods developed for hEtG determination published between years 2000 and 2017 is summarized in Tables 1a and 1b, where data on sample amount, details on analytical method and achieved sensitivity are reported for clarity [5–25,35,37–39]. Although GC–MS technique usually allows to reach lower LOQs also in presence of minor quantity of starting material, GC analysis requires a derivatization step, which contributes to increase complexity and renders the procedure time consuming. For this reason, probably, in recent years most of the
Corresponding author. E-mail address: [email protected] (J.P. Pascali).
https://doi.org/10.1016/j.jchromb.2018.09.027 Received 28 May 2018; Received in revised form 1 September 2018; Accepted 26 September 2018 Available online 29 September 2018 1570-0232/ © 2018 Elsevier B.V. All rights reserved.
Journal of Chromatography B 1100–1101 (2018) 33–38
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Table 1a Short overview of the available GC–MS methods for the determination of hEtG. Hair amount (mg)
Cut or ground
Clean up
Limit of quantification (pg/mg)
Technique
References
50 30 30 50 20 20 30 10–50 30
Ground Ground Ground Cut Cut Cut Ground Ground Ground
None Isolute NH2 SPE Oasis MAX SPE None Oasis MAX SPE Protein precipitation plate Oasis MAX SPE Cleanscreen EtG SPE SPE
5000 6 2.3 2.4 10 10 8.4 2.8 0.2
GC–MS EI GC–MS NICI GC–MS NICI GC–MS NICI GC–MS/MS EI GC–MS/MS EI GC–MS/MS NICI GC–MS/MS NICI GC–MS/MS NICI
[5] [6] [7] [8] [9] [10] [11] [12] [13]
Table 1b Short overview of the available LC-MS methods for the determination of hEtG. Hair amount (mg)
Cut or ground
Clean up
LC column chemistry
Limit of quantification (pg/mg)
Linearity (pg/mg)
References
100 100 50 100 30 25 100 50 100 50 30 25 50 75 30 50
Cut Cut Cut Ground Cut Cut Cut Cut Cut Cut Cut Ground Ground Ground Cut Ground
Isolute NH2 SPE None Oasis MAX SPE None Cleanscreen EtG SPE None None None Oasis MAX SPE Isolute NH2 SPE Cleanscreen EtG SPE Oasis MAX SPE BondElut SAX None Oasis MAX SPE None
phenyl-hexyl Synergy Polar-RP Chrompack Inertsil ODS-3 Acquity BEH HILIC Hypercarb Uptisphere-3SI Luna HILIC Sinergy Polar RP Acquity BEH C18 Inertsil ODS-3 Zorbax Eclipse XDB-C8 Acquity UPLC HSS T3 Hypercarb Acquity UPLC HSS T3 Hypercarb Synergi 4u fusion RP Acquity UPLC HSS T3
102 3 10 50 10 20 4 1 20 2.6 2 2 10 2.3 3 4.7
25–2000 3–2000 20–2000 50–5000 10–3000 20–1000 2–400 1–10 20–2500 2–200 2–330 2–100 10–500 4–400 3–500 n.d.
[14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [37] [35] [38] [39]
hEtG, exploiting this novel HILIC stationary phase and to discuss the obtained results in terms of sensitivity and sample pre-treatment convenience. The final method has been applied to real hair samples.
attention has been focused on LC-MS technique, which is easily prone to the analysis of more polar compounds in aqueous suspensions. However, many LC-MS methods exploit columns based on reversed-phase chemistry, requiring high amounts of aqueous mobile phase to retain polar compounds such as EtG, but strongly negatively affecting the ionization at MS source level. To overcome such limitation, postcolumn addition of acetonitrile is often used. In this frame, the introduction of more hydrophilic stationary phases for hEtG analysis have revealed successful since the work by Kintz and colleagues already in 2008 [16]. The term “hydrophilic interaction chromatography” was introduced by Alpert over 25 years ago to describe a liquid chromatography technique where polar or ionised solutes can be separated on a polar stationary phase with polar solvents containing water as a minor constituent of the mobile phase [26]. HILIC separations have undergone an upsurge in interest due to its numerous applications for the analysis of solutes of pharmaceutical, biomedical and clinical analysis, for which the technique is often suitable [27]. The advantages of HILIC include the ability to retain polar and ionic solutes that elute too readily in reversed-phase (RP) chromatography, offering an orthogonality in method development. For these reasons, applications of HILIC separation have been reviewed also recently in many scientific fields, such as pharmaceutical analysis [28], amino acids, peptides and proteins [29], proteomics [30] and metabolomics [31]. The broad range of applications is explained by the great variety of available bonded phases. In fact, HILIC phases can be divided into groups based on their chemical structure, which include neutral (e.g. amide, cyano, diol), positively charged (e.g. amino, imidazole, triazole), negatively charged (polyaspartic acid, bare silica) and zwitterionic (e.g. sulfobetaine or peptide) [32]. In this work, a proprietary zwitterionic stationary phase bonded on a sub 3 μm superficially porous particles has been adopted for the selective retention of ETG in hair matrix. The purpose of this study was to develop and validate an easy but sensitive method for the analysis of
2. Materials and methods 2.1. Materials EtG and the deuterated internal standard (d5-EtG) were obtained from Sigma Aldrich – Saint Louis, USA. Stock solution of EtG (1 mg/ml) was diluted in methanol to working standard solutions at concentrations 0,025–0.05–0,15–0,5 ng/μl. Diluted deuterated internal standard (d5-EtG) solution was prepared in methanol from the 0.1 mg/ml stock solution at the concentration of 0,250 ng/μl. All solutions were stored at −20 °C and left at room temperature at least 2 h for equilibration prior use. Methanol, acetonitrile and water for mobile phases preparation (all LC–MS grade) were purchased from Biosolve Chemie SARL – (Dieuze, France). Ammonium acetate was acquired from Carlo Erba Reagents (Milan, Italy). 2.2. Hair samples preparation All hair samples for analysis were collected from the vertex posterior region. From these samples a proximal 3 cm segment was used. For validation and analysis, 50 mg of hair were used and kept stored under dry conditions at room temperature until analysis. The selected hair segment was washed three times by shaking the sample with 20 ml of methanol for 5 min. The hair samples were subsequently left to dry at room temperature and then cut into small pieces. 50 mg of hair were weighted and 2500 pg d5-EtG as internal standard and 350 μl of water were added carefully to soak all the material. Samples were incubated overnight and then ultrasound extraction at 50 °C was applied for 2 h. The extract was collected, taken to dryness at 50 °C using a metal 34
Journal of Chromatography B 1100–1101 (2018) 33–38
D. Palumbo et al.
heating block then reconstituted in 150 μl water/acetonitrile, 10/90 (v/ v). For the comparative analysis of the present study, 12 real case hair samples were analysed with the present method and compared to an already validate method present in the literature [20]. From the 12 real cases hair samples, 3 were positive (EtG > 30 pg/mg) and 9 were negative (LOD < EtG < 30 pg/mg).
confidence) was determined by analysing seven replicates of the lowest point of calibration and by calculating the T-Students confidence at 99% interval. The LOQ was mathematically defined as equal to 10 times the standard deviation of the results for seven replicates at lowest concentration used to determine a justifiable limit of detection [34]. The RSD (%) at LOQ is typically determined < 20%. Both LOD and LOQ concentrations were experimentally verified in spiked hair matrix by using one sample for each hair type used for selectivity (no treatment, coloured, grey, thermal stressed hair).
2.3. Apparatus The LC MS system consisted of an Agilent 1290 high pressure liquidchromatography (HPLC) system coupled to an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA). EtG was separated employing an InfinityLab Poroshell 120 HILIC-Z column (3.0 × 100 mm, 2.7 μm), zwitterionic stationary phase on superficially porous particles (Agilent Technologies, Santa Clara, CA) at 30 °C. The mobile phase consisted of water added with 20 mM ammonium acetate pH 6.0 (A) and acetonitrile (B) with a flow rate of 0.3 ml/ min. The gradient was as follows: 90% B for 1 min, 90–80% B from 1 to 7 min, isocratic 80% B from 7 to 10 min, 80–90% B from 10 to 10.1 min followed by equilibration step of 5 min. During the method optimization phase, also an isocratic method at 85% B was developed and preliminary tested. The volume of injection was optimized and the final result was 5 μl. The triple quad instrument was operated in negative ion mode with capillary and nozzle voltages of 5000 V and 500 V, respectively, gas temperature at 325 °C, sheat gas temperature at 400 °C, nitrogen at 10 l/min as sheat gas and drying gas flows and nebulization at 40 psi. The analytes were detected in the multiple reaction monitoring mode (MRM), monitoring three transitions for EtG and one transitions for d5-EtG (Table 2). Analysis of the collected data was carried out with the Masshunther software (version B.04.00), (Agilent Technologies, Santa Clara, CA). All source parameters were optimized under LC conditions.
2.4.2. Precision, accuracy and matrix effect Precision and accuracy of the method were evaluated by measuring six replicates of QC samples at three different concentration levels (5, 40, 80 pg/mg) on three consecutive days. The matrix effect, expressed as bias %, was measured at three concentration levels (5, 30 and 100 pg/mg) by comparing the areas under the peaks from spiked hair (A) to spiked water at the same concentration (B) × 100 (Matrix effect = (A / B) ∗ 100). 2.4.3. Method application Method was verified by proficiency test samples routinely analysed in the lab (total number = 6) and applied to a set of real hair sample submitted for driving licence regranting, already analysed with the method reported in Ref. [20]. 3. Results Based on hydrophilic interaction chromatography a fast and reliable LC–MS/MS method was developed with optimized chromatographic separation for the quantification of hEtG achieving excellent retention in consideration of the polar nature of the molecule. The zwitterionic group of the stationary phase is in fact a N,N,N‑trimethyl taurine attached to a silane. The manufacturer used a proprietary bonding process to ensure that the surface does not have any cation or anion exchange (Fig. 2). Using this stationary phase, the retention times of hEtG were 7.2 min for the full validated gradient method and 5.2 min for the partially validated isocratic method. The calibration range was 5–100 pg/mg for both methods (weighted 1/x). The calculated LOD and LOQ were 1.4 and 4.5 pg/mg and 1.7 and 5 pg/mg, for the gradient and the isocratic method respectively. These limits are consistent with previously LC-MS published methods (Tables 1a and 1b) also in consideration of no sample purification step involved and any post-column addition of modifiers. Results for precision and accuracy are summarized in Table 3 for the gradient method with intraday and interday precision below 12% at LOQ and better than 10% for medium and high level. Accuracy, expressed as bias, was not > 7% for all levels. Good results were obtained also with the isocratic method (data not shown), however, full validation was decided for the gradient method because of better peak shape and better column preservation over time. The calculated matrix-related effects were 73% at 5 pg/mg (at LOQ), 87% at 30 pg/mg and 110% at 100 pg/mg (Table 4). The method, after preliminary verification by analysing proficiency test samples with satisfactory accuracies (average bias = 6%), was applied to a set of real hair sample of people of known alcohol consumption and/or from already analysed sample with the procedure
2.4. Method validation parameters Validation of the method was done according to Ref. [33]. The total run time for the gradient was 10 min and EtG eluted at 7.2 free of interfering peaks. A representative chromatogram of a hair extract spiked at 5 pg/mg EtG is shown in Fig. 1. The tested validation parameters were selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and matrix effect. 2.4.1. Selectivity, calibration model, LOD and LOQ The lack of response in blank matrix at retention time of EtG was assessed by analysing 15 different hair samples of teetotallers without any hair treatment, 7 samples from teetotallers with coloured hair, 2 samples from teetotallers with grey hair and 1 sample from teetotaller with thermal treated hair. Samples were analysed with and without IS addition. The calibration model was tested in spiked blank matrix at 4 calibration levels (0, 5, 10, 30 and 100 pg/mg), 6 replicates each level, by least-squares regression procedure estimation. Origin was not included and a weight factor of 1/x was applied. Limit of detection (LOD), defined as the lowest concentration level that can be determined to be statistically different from a blank (99% Table 2 MS parameters for the determination of EtG. Analyte a
EtG EtG EtG EtG-d5a a
Q1 (m/z)
Q2 (m/z)
Relative ion intensities and tolerance (%)
Dwell time (ms)
Collision energy (V)
Fragmentor (V)
Cell acceleration voltage (V)
221 221 221 226
75 85 113 85
100 98.9 ± 20 47.9 ± 20 100
120 120 120 120
12 8 12 8
90 90 90 96
5 7 3 7
Quantifier. 35
Journal of Chromatography B 1100–1101 (2018) 33–38
D. Palumbo et al.
EtG 3
EtG 2
EtG 1
I.S.
A
B
Fig. 1. LC–MS MRM-chromatogram of a hair extract spiked of EtG at 5 pg/mg and IS (A) and blank hair sample added of IS (B). MRM transitions: IS: 226/75, ETG 1: 221/75, EtG 2: 221/113 and EtG 3: 221/75.
poor homogeneity for the incorporated drugs both along the length and among different locks due to the mechanism of drugs incorporation. This could explain the different result obtained in one case (Fig. 3), where a discrepancy was observed between the two methods (positive vs negative). The two results, as well as all data, are obtained from independent extractions of aliquots of the same sample. 4. Discussion Some recent studies have described a shift from traditional reversedphase to more hydrophilic LC chemistry for hEtG determination. The reason relies on the poor retention of C8 and C18 columns for polar compounds, even in presence of great amount of aqueous phase. So far, porous graphitic carbon column has been suggested to give better retention of EtG and better separation from the hair matrix [35]. However, to reach satisfactory diagnostic sensitivity, a post-column addition of solvent had to be introduced to improve EtG ionization at source level. Moreover, digested hair matrix usually produces important signal suppression, due to the intrinsic ions and biological compounds released in solution, thus a sample purification step is usually introduced. With the introduction of zwitterionic HILIC chemistry, which is based on complex multiparametric retention processes of partitioning, adsorption and eventually electrostatic interactions between the analytes and the column constituents, some of these problems seems to be, at least, partially resolved for hEtG determination. In fact, the developed method, based on a separation at neutral conditions confirms the optimum retention and thus selectivity for weak acids in zwitterionic column [36]. Such good performances are ascribed to increased EtG ionization at pH 6 and retention by hydrophilic/hydrogen bonding processes at stationary phase level. Furthermore, HILIC strategy, allowing the elution of polar analytes with high amounts of acetonitrile, is suitable for in source ionization and thus to MS-coupling. In fact, in the present method, EtG eluted at 80% acetonitrile, avoiding the need for post-column solvent addition. Data obtained from validation tests were in good agreement with previously published analytical methods (see Table 1a and 1b), both in terms of sensitivity and accuracy, also in consideration of the faster and easier sample preparation and the absence of post-column acetonitrile addition. Similarly, matrix presence in the extracts, usually considered a drawback in LC-MS analysis for creating signal suppression/enhancement, does not seem to affect greatly the analysis. In conclusion, zwitterionic HILIC stationary phase may add an important contribution to the analysis of tricky weak acids
Fig. 2. A schematic representation of the zwitterionic group of the stationary phase of the HILIC-Z column is depicted. Table 3 Validation data for precision and accuracy.
RSD (%) intraday precision (n = 6) RSD (%) interday precision (n = 18) Accuracy (bias%)
EtG 5 pg/ mg
EtG 40 pg/mg
EtG 80 pg/mg
10.8
7.3
2.0
12.0
9.4
3.2
7
4
1
Table 4 Validation data for matrix effect and recovery.
Matrix effect (%) (RSD%) (n = 6)
EtG 5 pg/mg
EtG 30 pg/mg
EtG 100 pg/mg
73 (12)
87 (13)
110 (8)
reported in the Ref. [20], data results are summarized in Fig. 3. Although the number of samples could be increased in the future, the obtained results are in good agreement (R2 = 0.777) also in consideration of the analysed sample. In fact, on one hand hair is a strong, stable tissue less affected by adulterants and shows advantages over traditional matrices (i.e. blood, urine), on the other hand it suffers of 36
Journal of Chromatography B 1100–1101 (2018) 33–38
D. Palumbo et al.
Methods comparison 70.00 60.00
Ref. [20]
50.00 40.00 30.00 20.00 10.00 0.00 0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Current method
Fig. 3. Graphical comparison of the hEtG results obtained with the current method and an already validated and published method.
in biological matrices, such as hEtG, improving separation of analytes from matrix components. [13]
5. Conclusions A fast and reliable LC–MS/MS method, based on a zwitterionic stationary phase on 2.7 μm superficially porous particles was developed and validated. The method showed suitable for the diagnostic determination of hEtG in terms of sensitivity, precision and accuracy.
[14] [15]
Funding
[16]
None.
[17]
Acknowledgements
[18]
None. References
[19]
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Journal of Chromatography B 1100–1101 (2018) 33–38
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Novel zwitterionic HILIC stationary phase for the determination of ethyl glucuronide in human hair by LC-MS/MS
T
Diego Palumboa, Paolo Faisb, Anna Calìc, Monica Lusardic, Elisabetta Bertola, ⁎ Jennifer P. Pascalia, a
Forensic Toxicology Division, Department of Health Sciences, University of Florence, Largo Brambilla 3, 50134 Florence, Italy University of Bologna, Department of Medical and Surgical Sciences, Unit of Legal Medicine, Via Irnerio 49, Bologna, Italy c Agilent Technologies, Via V. Lamaro, Rome, Italy b
ARTICLE INFO
ABSTRACT
Keywords: Ethyl glucuronide Liquid chromatography Hair Hydrophilic chromatography Alcohol HILIC-Z
Some recent studies have described a shift from traditional reversed-phase to more hydrophilic LC chemistry for EtG determination in hair (hEtG). The reason relies on the poor retention of C8– and C18-based columns for polar compounds, even in presence of great amount of aqueous phase. This work presents the development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-LC-MS/MS) method based on a novel zwitterionic stationary phase for the analysis of hEtG. The linearity was assessed in the range of 5–100 pg/mg hair, with a correlation coefficient of > 0.99. The method was selective and sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1.4 pg/mg and 4.5 pg/mg in hair, respectively. Suitable diagnostic sensitivity was achieved without the introduction of a sample purification step, or a post column solvent addition. The method was successfully applied to real hair samples after full validation. This method, based on a separation at neutral conditions, confirmed the optimum retention and thus selectivity for weak acids in zwitterionic HILIC columns.
1. Introduction Alcohol is the most widely consumed psychoactive substance and is becoming a problematic addiction issue in millions of people worldwide. In fact, unhealthy alcohol use can be either a primary or a secondary cause of liver disease and besides medical complications its abuse can be responsible of severe social problems [1,2]. Additionally, from the forensic point of view, it is of extreme importance to monitor alcohol abstinence in patients undergoing an alcohol withdrawal treatment when a legal cause is undergoing. Laboratory testing traditionally based diagnosis on the assessment of biomarkers such as mean corpuscular volume (MCV), carbohydrate deficient transferrin (CDT) and liver enzymes (gamma glutamyl-transferase, aspartate aminotransferase and alanine aminotransferase, aspartate-amine-transferase and alanine-amine-transferase). In the last few years, increased attention has been paid to ethyl glucuronide (EtG), a new specific alcohol intake marker [3]. It is a direct metabolite of non-oxidative breakdown of ethanol accounting for < 0.1% of total ethanol elimination. Through the measurement of EtG in hair (hEtG), it is possible to assess both the chronic alcohol abuse over time and to document the treatment efficacy of patients in a withdrawal program. However, due to its high polarity, ⁎
EtG is incorporated into hair only in very small amounts and thus the reliable detection of EtG in hair requires sensitive techniques in reason of the low concentrations (pg/mg range) finally present in the matrix, even in presence of abuse behaviours. Previous researches have shown that analytical methods based on gas chromatography (GC) with mass spectrometry (MS) and liquid chromatography (LC) with MS are the techniques of choice and, to reach appropriate sensitivity, sample preparation based on solid-phase extraction (SPE) is, in most cases, applied to obtain cleaner extracts. The recommended cut-off levels for hEtG diagnostic purposes, suggested by the Society of Hair Testing (SoHT), are substantially two values: 30 pg/mg to distinguish from moderate and heavy alcohol consumption and below 7 pg/mg to pass the medical assessment of alcohol abstinence [4]. A small overview on analytical methods developed for hEtG determination published between years 2000 and 2017 is summarized in Tables 1a and 1b, where data on sample amount, details on analytical method and achieved sensitivity are reported for clarity [5–25,35,37–39]. Although GC–MS technique usually allows to reach lower LOQs also in presence of minor quantity of starting material, GC analysis requires a derivatization step, which contributes to increase complexity and renders the procedure time consuming. For this reason, probably, in recent years most of the
Corresponding author. E-mail address: [email protected] (J.P. Pascali).
https://doi.org/10.1016/j.jchromb.2018.09.027 Received 28 May 2018; Received in revised form 1 September 2018; Accepted 26 September 2018 Available online 29 September 2018 1570-0232/ © 2018 Elsevier B.V. All rights reserved.
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Table 1a Short overview of the available GC–MS methods for the determination of hEtG. Hair amount (mg)
Cut or ground
Clean up
Limit of quantification (pg/mg)
Technique
References
50 30 30 50 20 20 30 10–50 30
Ground Ground Ground Cut Cut Cut Ground Ground Ground
None Isolute NH2 SPE Oasis MAX SPE None Oasis MAX SPE Protein precipitation plate Oasis MAX SPE Cleanscreen EtG SPE SPE
5000 6 2.3 2.4 10 10 8.4 2.8 0.2
GC–MS EI GC–MS NICI GC–MS NICI GC–MS NICI GC–MS/MS EI GC–MS/MS EI GC–MS/MS NICI GC–MS/MS NICI GC–MS/MS NICI
[5] [6] [7] [8] [9] [10] [11] [12] [13]
Table 1b Short overview of the available LC-MS methods for the determination of hEtG. Hair amount (mg)
Cut or ground
Clean up
LC column chemistry
Limit of quantification (pg/mg)
Linearity (pg/mg)
References
100 100 50 100 30 25 100 50 100 50 30 25 50 75 30 50
Cut Cut Cut Ground Cut Cut Cut Cut Cut Cut Cut Ground Ground Ground Cut Ground
Isolute NH2 SPE None Oasis MAX SPE None Cleanscreen EtG SPE None None None Oasis MAX SPE Isolute NH2 SPE Cleanscreen EtG SPE Oasis MAX SPE BondElut SAX None Oasis MAX SPE None
phenyl-hexyl Synergy Polar-RP Chrompack Inertsil ODS-3 Acquity BEH HILIC Hypercarb Uptisphere-3SI Luna HILIC Sinergy Polar RP Acquity BEH C18 Inertsil ODS-3 Zorbax Eclipse XDB-C8 Acquity UPLC HSS T3 Hypercarb Acquity UPLC HSS T3 Hypercarb Synergi 4u fusion RP Acquity UPLC HSS T3
102 3 10 50 10 20 4 1 20 2.6 2 2 10 2.3 3 4.7
25–2000 3–2000 20–2000 50–5000 10–3000 20–1000 2–400 1–10 20–2500 2–200 2–330 2–100 10–500 4–400 3–500 n.d.
[14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [37] [35] [38] [39]
hEtG, exploiting this novel HILIC stationary phase and to discuss the obtained results in terms of sensitivity and sample pre-treatment convenience. The final method has been applied to real hair samples.
attention has been focused on LC-MS technique, which is easily prone to the analysis of more polar compounds in aqueous suspensions. However, many LC-MS methods exploit columns based on reversed-phase chemistry, requiring high amounts of aqueous mobile phase to retain polar compounds such as EtG, but strongly negatively affecting the ionization at MS source level. To overcome such limitation, postcolumn addition of acetonitrile is often used. In this frame, the introduction of more hydrophilic stationary phases for hEtG analysis have revealed successful since the work by Kintz and colleagues already in 2008 [16]. The term “hydrophilic interaction chromatography” was introduced by Alpert over 25 years ago to describe a liquid chromatography technique where polar or ionised solutes can be separated on a polar stationary phase with polar solvents containing water as a minor constituent of the mobile phase [26]. HILIC separations have undergone an upsurge in interest due to its numerous applications for the analysis of solutes of pharmaceutical, biomedical and clinical analysis, for which the technique is often suitable [27]. The advantages of HILIC include the ability to retain polar and ionic solutes that elute too readily in reversed-phase (RP) chromatography, offering an orthogonality in method development. For these reasons, applications of HILIC separation have been reviewed also recently in many scientific fields, such as pharmaceutical analysis [28], amino acids, peptides and proteins [29], proteomics [30] and metabolomics [31]. The broad range of applications is explained by the great variety of available bonded phases. In fact, HILIC phases can be divided into groups based on their chemical structure, which include neutral (e.g. amide, cyano, diol), positively charged (e.g. amino, imidazole, triazole), negatively charged (polyaspartic acid, bare silica) and zwitterionic (e.g. sulfobetaine or peptide) [32]. In this work, a proprietary zwitterionic stationary phase bonded on a sub 3 μm superficially porous particles has been adopted for the selective retention of ETG in hair matrix. The purpose of this study was to develop and validate an easy but sensitive method for the analysis of
2. Materials and methods 2.1. Materials EtG and the deuterated internal standard (d5-EtG) were obtained from Sigma Aldrich – Saint Louis, USA. Stock solution of EtG (1 mg/ml) was diluted in methanol to working standard solutions at concentrations 0,025–0.05–0,15–0,5 ng/μl. Diluted deuterated internal standard (d5-EtG) solution was prepared in methanol from the 0.1 mg/ml stock solution at the concentration of 0,250 ng/μl. All solutions were stored at −20 °C and left at room temperature at least 2 h for equilibration prior use. Methanol, acetonitrile and water for mobile phases preparation (all LC–MS grade) were purchased from Biosolve Chemie SARL – (Dieuze, France). Ammonium acetate was acquired from Carlo Erba Reagents (Milan, Italy). 2.2. Hair samples preparation All hair samples for analysis were collected from the vertex posterior region. From these samples a proximal 3 cm segment was used. For validation and analysis, 50 mg of hair were used and kept stored under dry conditions at room temperature until analysis. The selected hair segment was washed three times by shaking the sample with 20 ml of methanol for 5 min. The hair samples were subsequently left to dry at room temperature and then cut into small pieces. 50 mg of hair were weighted and 2500 pg d5-EtG as internal standard and 350 μl of water were added carefully to soak all the material. Samples were incubated overnight and then ultrasound extraction at 50 °C was applied for 2 h. The extract was collected, taken to dryness at 50 °C using a metal 34
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D. Palumbo et al.
heating block then reconstituted in 150 μl water/acetonitrile, 10/90 (v/ v). For the comparative analysis of the present study, 12 real case hair samples were analysed with the present method and compared to an already validate method present in the literature [20]. From the 12 real cases hair samples, 3 were positive (EtG > 30 pg/mg) and 9 were negative (LOD < EtG < 30 pg/mg).
confidence) was determined by analysing seven replicates of the lowest point of calibration and by calculating the T-Students confidence at 99% interval. The LOQ was mathematically defined as equal to 10 times the standard deviation of the results for seven replicates at lowest concentration used to determine a justifiable limit of detection [34]. The RSD (%) at LOQ is typically determined < 20%. Both LOD and LOQ concentrations were experimentally verified in spiked hair matrix by using one sample for each hair type used for selectivity (no treatment, coloured, grey, thermal stressed hair).
2.3. Apparatus The LC MS system consisted of an Agilent 1290 high pressure liquidchromatography (HPLC) system coupled to an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA). EtG was separated employing an InfinityLab Poroshell 120 HILIC-Z column (3.0 × 100 mm, 2.7 μm), zwitterionic stationary phase on superficially porous particles (Agilent Technologies, Santa Clara, CA) at 30 °C. The mobile phase consisted of water added with 20 mM ammonium acetate pH 6.0 (A) and acetonitrile (B) with a flow rate of 0.3 ml/ min. The gradient was as follows: 90% B for 1 min, 90–80% B from 1 to 7 min, isocratic 80% B from 7 to 10 min, 80–90% B from 10 to 10.1 min followed by equilibration step of 5 min. During the method optimization phase, also an isocratic method at 85% B was developed and preliminary tested. The volume of injection was optimized and the final result was 5 μl. The triple quad instrument was operated in negative ion mode with capillary and nozzle voltages of 5000 V and 500 V, respectively, gas temperature at 325 °C, sheat gas temperature at 400 °C, nitrogen at 10 l/min as sheat gas and drying gas flows and nebulization at 40 psi. The analytes were detected in the multiple reaction monitoring mode (MRM), monitoring three transitions for EtG and one transitions for d5-EtG (Table 2). Analysis of the collected data was carried out with the Masshunther software (version B.04.00), (Agilent Technologies, Santa Clara, CA). All source parameters were optimized under LC conditions.
2.4.2. Precision, accuracy and matrix effect Precision and accuracy of the method were evaluated by measuring six replicates of QC samples at three different concentration levels (5, 40, 80 pg/mg) on three consecutive days. The matrix effect, expressed as bias %, was measured at three concentration levels (5, 30 and 100 pg/mg) by comparing the areas under the peaks from spiked hair (A) to spiked water at the same concentration (B) × 100 (Matrix effect = (A / B) ∗ 100). 2.4.3. Method application Method was verified by proficiency test samples routinely analysed in the lab (total number = 6) and applied to a set of real hair sample submitted for driving licence regranting, already analysed with the method reported in Ref. [20]. 3. Results Based on hydrophilic interaction chromatography a fast and reliable LC–MS/MS method was developed with optimized chromatographic separation for the quantification of hEtG achieving excellent retention in consideration of the polar nature of the molecule. The zwitterionic group of the stationary phase is in fact a N,N,N‑trimethyl taurine attached to a silane. The manufacturer used a proprietary bonding process to ensure that the surface does not have any cation or anion exchange (Fig. 2). Using this stationary phase, the retention times of hEtG were 7.2 min for the full validated gradient method and 5.2 min for the partially validated isocratic method. The calibration range was 5–100 pg/mg for both methods (weighted 1/x). The calculated LOD and LOQ were 1.4 and 4.5 pg/mg and 1.7 and 5 pg/mg, for the gradient and the isocratic method respectively. These limits are consistent with previously LC-MS published methods (Tables 1a and 1b) also in consideration of no sample purification step involved and any post-column addition of modifiers. Results for precision and accuracy are summarized in Table 3 for the gradient method with intraday and interday precision below 12% at LOQ and better than 10% for medium and high level. Accuracy, expressed as bias, was not > 7% for all levels. Good results were obtained also with the isocratic method (data not shown), however, full validation was decided for the gradient method because of better peak shape and better column preservation over time. The calculated matrix-related effects were 73% at 5 pg/mg (at LOQ), 87% at 30 pg/mg and 110% at 100 pg/mg (Table 4). The method, after preliminary verification by analysing proficiency test samples with satisfactory accuracies (average bias = 6%), was applied to a set of real hair sample of people of known alcohol consumption and/or from already analysed sample with the procedure
2.4. Method validation parameters Validation of the method was done according to Ref. [33]. The total run time for the gradient was 10 min and EtG eluted at 7.2 free of interfering peaks. A representative chromatogram of a hair extract spiked at 5 pg/mg EtG is shown in Fig. 1. The tested validation parameters were selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and matrix effect. 2.4.1. Selectivity, calibration model, LOD and LOQ The lack of response in blank matrix at retention time of EtG was assessed by analysing 15 different hair samples of teetotallers without any hair treatment, 7 samples from teetotallers with coloured hair, 2 samples from teetotallers with grey hair and 1 sample from teetotaller with thermal treated hair. Samples were analysed with and without IS addition. The calibration model was tested in spiked blank matrix at 4 calibration levels (0, 5, 10, 30 and 100 pg/mg), 6 replicates each level, by least-squares regression procedure estimation. Origin was not included and a weight factor of 1/x was applied. Limit of detection (LOD), defined as the lowest concentration level that can be determined to be statistically different from a blank (99% Table 2 MS parameters for the determination of EtG. Analyte a
EtG EtG EtG EtG-d5a a
Q1 (m/z)
Q2 (m/z)
Relative ion intensities and tolerance (%)
Dwell time (ms)
Collision energy (V)
Fragmentor (V)
Cell acceleration voltage (V)
221 221 221 226
75 85 113 85
100 98.9 ± 20 47.9 ± 20 100
120 120 120 120
12 8 12 8
90 90 90 96
5 7 3 7
Quantifier. 35
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D. Palumbo et al.
EtG 3
EtG 2
EtG 1
I.S.
A
B
Fig. 1. LC–MS MRM-chromatogram of a hair extract spiked of EtG at 5 pg/mg and IS (A) and blank hair sample added of IS (B). MRM transitions: IS: 226/75, ETG 1: 221/75, EtG 2: 221/113 and EtG 3: 221/75.
poor homogeneity for the incorporated drugs both along the length and among different locks due to the mechanism of drugs incorporation. This could explain the different result obtained in one case (Fig. 3), where a discrepancy was observed between the two methods (positive vs negative). The two results, as well as all data, are obtained from independent extractions of aliquots of the same sample. 4. Discussion Some recent studies have described a shift from traditional reversedphase to more hydrophilic LC chemistry for hEtG determination. The reason relies on the poor retention of C8 and C18 columns for polar compounds, even in presence of great amount of aqueous phase. So far, porous graphitic carbon column has been suggested to give better retention of EtG and better separation from the hair matrix [35]. However, to reach satisfactory diagnostic sensitivity, a post-column addition of solvent had to be introduced to improve EtG ionization at source level. Moreover, digested hair matrix usually produces important signal suppression, due to the intrinsic ions and biological compounds released in solution, thus a sample purification step is usually introduced. With the introduction of zwitterionic HILIC chemistry, which is based on complex multiparametric retention processes of partitioning, adsorption and eventually electrostatic interactions between the analytes and the column constituents, some of these problems seems to be, at least, partially resolved for hEtG determination. In fact, the developed method, based on a separation at neutral conditions confirms the optimum retention and thus selectivity for weak acids in zwitterionic column [36]. Such good performances are ascribed to increased EtG ionization at pH 6 and retention by hydrophilic/hydrogen bonding processes at stationary phase level. Furthermore, HILIC strategy, allowing the elution of polar analytes with high amounts of acetonitrile, is suitable for in source ionization and thus to MS-coupling. In fact, in the present method, EtG eluted at 80% acetonitrile, avoiding the need for post-column solvent addition. Data obtained from validation tests were in good agreement with previously published analytical methods (see Table 1a and 1b), both in terms of sensitivity and accuracy, also in consideration of the faster and easier sample preparation and the absence of post-column acetonitrile addition. Similarly, matrix presence in the extracts, usually considered a drawback in LC-MS analysis for creating signal suppression/enhancement, does not seem to affect greatly the analysis. In conclusion, zwitterionic HILIC stationary phase may add an important contribution to the analysis of tricky weak acids
Fig. 2. A schematic representation of the zwitterionic group of the stationary phase of the HILIC-Z column is depicted. Table 3 Validation data for precision and accuracy.
RSD (%) intraday precision (n = 6) RSD (%) interday precision (n = 18) Accuracy (bias%)
EtG 5 pg/ mg
EtG 40 pg/mg
EtG 80 pg/mg
10.8
7.3
2.0
12.0
9.4
3.2
7
4
1
Table 4 Validation data for matrix effect and recovery.
Matrix effect (%) (RSD%) (n = 6)
EtG 5 pg/mg
EtG 30 pg/mg
EtG 100 pg/mg
73 (12)
87 (13)
110 (8)
reported in the Ref. [20], data results are summarized in Fig. 3. Although the number of samples could be increased in the future, the obtained results are in good agreement (R2 = 0.777) also in consideration of the analysed sample. In fact, on one hand hair is a strong, stable tissue less affected by adulterants and shows advantages over traditional matrices (i.e. blood, urine), on the other hand it suffers of 36
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Methods comparison 70.00 60.00
Ref. [20]
50.00 40.00 30.00 20.00 10.00 0.00 0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Current method
Fig. 3. Graphical comparison of the hEtG results obtained with the current method and an already validated and published method.
in biological matrices, such as hEtG, improving separation of analytes from matrix components. [13]
5. Conclusions A fast and reliable LC–MS/MS method, based on a zwitterionic stationary phase on 2.7 μm superficially porous particles was developed and validated. The method showed suitable for the diagnostic determination of hEtG in terms of sensitivity, precision and accuracy.
[14] [15]
Funding
[16]
None.
[17]
Acknowledgements
[18]
None. References
[19]
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