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Identification and quantitation of phosphatidylethanols in oral fluid by liquid chromatography-tandem mass spectrometry

  • Shahid Ullah EMAIL logo , Anders Helander and Olof Beck
Published/Copyright: December 19, 2016
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 55 Issue 9

Abstract

Background:

Phosphatidylethanols (PEth) are formed from phosphatidylcholines and ethanol and are used as a specific and sensitive alcohol biomarker. An analytical method for analysis of PEth in oral fluid based on high-performance liquid chromatography coupled to a quadrupole tandem mass spectrometer (LC-MS/MS) was developed and validated and applied on samples collected from patients undergoing alcohol detoxification.

Methods:

A 200-μL aliquot of oral fluid, collected using the QuantisalTM device, was extracted with chloroform/methanol containing internal standard and subjected to LC-MS/MS analysis of three selected PEth forms (16:0/16:0, 16:0/18:1, and 16:0/18:2). Chromatographic separation was achieved on a UPLC BEH phenyl column, using a mobile phase consisting of acetonitrile and water containing 10 mmol/L ammonium hydrogen carbonate with 0.1% ammonia. The MS instrument was operated in negative electrospray ionization and selected reaction monitoring mode.

Results:

The detection limit for PEth 16:0/16:0, 16:0/18:1, and 16:0/18:2 was ~0.1 ng/mL, and the extraction recoveries at 2.0 ng/mL were in the range of 99%–114%. Method linearity over a concentration range up to 200 ng/mL was ≥0.99. No significant deviation in results was observed in an analyte stability study of two different concentrations at two different temperatures over 3 months. In 35 oral fluid samples collected from patients undergoing alcohol detoxification, the highest concentration was observed for PEth 16:0/18:1 (Detected range, 0.51–55.3 ng/mL; mean, 8.5; median, 3.1). In addition, all three PEth forms were variably identified in a majority (63%) of the oral fluid samples. The PEth 16:0/18:1 values in oral fluid showed a weak positive correlation with the corresponding values in whole blood samples (r=0.50, p=0.026, n=20).

Conclusions:

The LC-MS/MS method could reliably detect and quantify PEth in oral fluid samples collected after alcohol exposure. The method was characterized by validation data with satisfactory recovery, sensitivity, accuracy, and imprecision, and applied for analysis of clinical samples. The results suggest that measurement of PEth in oral fluid can be used as a biomarker for alcohol consumption, and as such a non-invasive complement to analysis in blood. However, further studies are required to evaluate the test characteristics (e.g. sensitivity and half-life) in comparison with PEth in blood.

Keywords: oral fluid; phosphatidylethanol (PEth) 16:0/18:1; quadrupole tandem mass spectrometer (LC-MS/MS)

Introduction

Measurement of phosphatidylethanols (PEth) in whole blood samples is used as a specific biomarker of recent, excessive alcohol consumption and as an objective indicator to support alcohol abstinence. The formation of PEth results from a transphosphatidylation reaction from the corresponding phosphatidylcholine precursor in the presence of ethanol, by action of the enzyme phospholipase D [1], [2]. Due to the large number of phosphatidylcholines, PEth also consists of a large number of molecular subforms based on a common glycerol backbone, a polar head group, and two fatty acid moieties of variable length and number of double bonds.

The presence of PEth in whole blood samples after alcohol exposure has been reported in several studies [3], [4], [5], [6], [7], with PEth 16:0/18:1 and 16:0/18:2 typically being the predominant forms [1]. The formation of PEth is indicated to be ethanol-dose dependent [8], [9] and it remains detectable in blood for several weeks of abstinence after a period of prolonged heavy drinking [10], [11], [12]. Being a phospholipid, PEth is present in cell membranes and in the blood, mainly found in the erythrocytes [13]. Although blood sampling is a routine clinical practice, the collection of venous blood is invasive, requires skilled personnel and suitable sampling premises, and is sometimes problematic due to patient’s feeling of inconvenience. PEth can also be measured in dried blood samples [14], [15], which may be advantageous due to the smaller sample volume required (e.g. finger-prick blood) and improved stability on storage [16], [17].

Because PEth is most likely formed in all cell membranes of the body [18], alternative biological matrices containing phosphatidylcholines may be used for PEth testing. For example, a recent study emphasized that PEth was detectable in exhaled breath particles, which have been demonstrated to contain phosphatidylcholines [19], after chronic heavy drinking [20].

In recent years, oral fluid (“saliva”) has become an established alternative, non-invasive, and readily available specimen for drug testing [21], [22], [23]. Phosphatidylcholines were recently found to be present in oral fluid [18], suggesting that PEth also may be detectable in this matrix.

The present study was undertaken to evaluate oral fluid as a possible sample for analysis of PEth as an alcohol biomarker. An analytical method for identification and quantification of 3 PEth forms in oral fluid, based on high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer (LC-MS/MS), was developed and validated and applied on samples collected from patients undergoing alcohol detoxification. The PEth forms selected for this study (PEth 16:0/16:0, 16:0/18:1, and 16:0/18:2) were chosen because these are among the most predominant forms in human blood [1], [5], [17].

Materials and methods

Chemicals

Reference compounds for 1,2-dipalmitoyl-sn-glycero-3-phosphoethanol (PEth 16:0/16:0), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanol (PEth 16:0/18:2), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol-d31 (PEth 16:0/18:1-d31; internal standard, IS) were obtained from Avanti Polar Lipids Co (Alabaster, AL, USA), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (PEth 16:0/18:1) was from MedChem101 LLC (Comshohocken, PA, USA).

LC-MS-grade methanol and acetonitrile were purchased from Fisher Scientific AB (Gothenburg, Sweden), ammonium hydrogen carbonate from VWR (Leuven, Belgium), and ammonia (25%) from Merck KGaA (Darmstadt, Germany). Ultrapure water (>18 MΩ/cm) was prepared in-house with a Milli-Q water purification system (Merck Millipore, Solna, Sweden).

Oral fluid collection and sample treatment

Collection of oral fluid was performed using a commercial liquid based collection device (QuantisalTM, Immunalysis, Pomona, CA, USA). In this system, approximately 1 mL of oral fluid is sampled in 3 mL of stabilizing buffer. The samples were stored at −20 °C until analysis.

Blank samples of oral fluid in Quantisal buffer to be used in the method development were collected from 20 healthy volunteers (“social drinkers” and teetotalers, according to self-report) at the Karolinska University Laboratory Huddinge (Stockholm, Sweden). The social drinkers reported typically consuming <5 glasses of wine, or corresponding amounts of other beverages, per month and had not been drinking alcohol for at least 3 days prior to sampling.

A Folch extraction [24] of the oral fluid samples was performed prior to the analysis of PEth. Briefly, a 200-μL aliquot of the oral fluid in Quantisal buffer sample was transferred to a glass tube, and 400 μL chloroform and 200 μL methanol containing IS were added. After vortexing for 30 s, the sample was centrifuged for 5 min at 2500 rpm. The lower part, chloroform phase, was transferred to another glass tube and evaporated to dryness, followed by reconstitution with 70 μL methanol:0.1% ammonia in water (75%:25%, v/v).

Calibration standards and quality control samples

All reference substances were received in powder form. Stock solutions were prepared in chloroform (10 mg/mL for PEth 16:0/16:0, PEth 16:0/18:1, and PEth 16:0/18:1-d31, and 2.5 mg/mL for PEth 16:0/18:2) and further diluted with methanol to working solutions (400 μg/mL for PEth 16:0/16:0 and 16:0/18:1, 50 μg/mL for 16:0/18:2, and 500 μg/mL for 16:0/18:1-d31). The working solutions were stored at −20 °C

Final calibrators for daily use covering the concentration range from the individual lower limit of quantification (LLOQ) to 200 ng/mL were prepared in blank samples (oral fluid in Quantisal buffer) by spiking with working solutions. Quality control samples for all PEth forms at low (1 ng/mL) and high (10 ng/mL) concentrations were prepared in blank samples by spiking with working solutions.

Instrumentation

The LC-MS/MS instrumentation consisted of a Thermo Fisher Scientific Dionex Ultima 3000 UHPLC system with a RS binary solvent pump, column oven and RS auto sampler. The mass spectrometer was a TSQ Quantiva triple quadrupole (Thermo). The system was operated using Chromeleon Dionex chromatography mass spectrometry link 2.14, Trace Finder Clinical Research version 3.2, and Thermo TSQ Quantiva tune application 1.1 software. The heated electrospray ionization (HESI) was operated in negative ion mode. The following conditions were used in the mass spectrometer: ion spray voltage, 3.2 kV; vaporizer temperature, 350 °C; ion transfer tube temperature, 280 °C; peak width, 0.70 FWHM for Q1 and Q3 resolution. Ion intensities were recorded in selected ion monitoring mode with a dwell time 0.05 s. During method optimization, the parent-to-product ion transitions monitored were m/z 675.3>255.2 (quantifier) and 437.2 for PEth 16:0/16:0, m/z 701.6>255.1 (quantifier) and 281.2 for PEth 16:0/18:1, m/z 699.5>255.3 (quantifier) and 279.2 for PEth 16:0/18:2, and m/z 731.6>281.2 for PEth 16:0/18:1-d31. For PEth 16:0/18:1, m/z 255.1 was chosen as the quantification ion, due to an analytical interference at m/z 281.2 in oral fluid samples.

Chromatographic separation was performed using an Acquity BEH (ethylene bridged hybrid) phenyl column (50 mm×1.0 mm, 1.7 μm; Waters) connected with a Vanguard pre-column (5×2.1 mm, 1.7 μm; Waters). The chromatographic system was operated in gradient mode with flow rate of 200 μL/min. Mobile phase A consisted of methanol/water (5%:95%, v/v) containing 10 mmol/L ammonium hydrogen carbonate and 0.1% ammonia, and mobile phase B consisted of 100% acetonitrile. The gradient settings were 0–0.3 min 25% B, 0.3–0.5 min linear change to 48% B, 0.5–2.5 min linear change to 77% B, and 2.5–3.5 min linear change to 98% B, 3.5–4.5 min 98% B, and 4.5–5.0 min back to 25% B for recondition. The column oven temperature was set at 40 °C.

A 2.0-μL sample volume was injected. A strong needle wash with water/methanol/acetonitrile/isopropyl alcohol (25% each) was performed before and after each injection. With this setting, nice chromatograms were achieved for PEth in spiked blank and patient oral fluid samples, as demonstrated in Figure 1.

Figure 1: Representative chromatograms from the LC-MS/MS analysis of 3 PEth forms (16:0/16:0, 16:0/18:1, and 16:0/18:2) in blank oral fluid (A, B, C), spiked blank oral fluid (0.4 ng/mL; D, E, F) and a patient sample (G, H, I).The PEth concentrations in the patient sample were 1.3 ng/mL for PEth 16:0/16:0, 15.1 ng/mL for PEth 16:0/18:1, and 3.9 ng/mL for PEth 16:0/18:2.
Figure 1:

Representative chromatograms from the LC-MS/MS analysis of 3 PEth forms (16:0/16:0, 16:0/18:1, and 16:0/18:2) in blank oral fluid (A, B, C), spiked blank oral fluid (0.4 ng/mL; D, E, F) and a patient sample (G, H, I).

The PEth concentrations in the patient sample were 1.3 ng/mL for PEth 16:0/16:0, 15.1 ng/mL for PEth 16:0/18:1, and 3.9 ng/mL for PEth 16:0/18:2.

Method validation

The LC-MS/MS method for PEth in oral fluid was validated by determining the lower detection limit (LOD), LLOQ, linearity, imprecision and accuracy, recovery, interferences, matrix effect, instrument carry-over, and stability of samples on storage. All validation experiments were performed using blank oral fluid samples in Quantisal buffer. Measured values were calculated back to the actual concentration in neat oral fluid, by correction for the dilution factor during sampling (i.e. measured values×4).

The LOD and LLOQ were determined in the chromatograms from 10 aliquots of blank oral fluid samples from 10 subjects spiked with 10 pg of each PEth form in each aliquot (0.2 ng/mL). The LOD and LLOQ were calculated from the acquired values and corresponding sample chromatograms based on signal-to-noise ratios of 3 and 10, respectively. The achieved LLOQ was determined based on a total coefficient of variation (CV) <20% and the 80%–120% accuracy criterion.

Method linearity was documented over a concentration range from the individual LLOQ up to 200 ng/mL, by spiking blank oral fluid samples at six concentrations. Method imprecision and accuracy were evaluated by spiking five replicates of blank samples at two concentrations (0.4 ng/mL and 20 ng/mL) over 5 days (n=25).

The recovery of PEth was determined from three blank oral fluid samples spiked with 2.0 ng/mL of each form before and after extraction. In both cases, the IS was added after extraction during reconstitution.

Interference experiments were performed using oral fluid samples collected from 20 healthy volunteers (social drinkers and teetotalers). Matrix effect was evaluated in two ways; first by post column infusion of 1.0 μg/mL of each PEth in methanol:0.1% ammonia solution (75%:25%, v/v) at a constant flow rate of 10 μL/min. Blank extracts from a healthy volunteer and methanol were injected simultaneously into the LC, while monitoring the selected SRM transitions for all PEth forms. In a second experiment, three replicates of blank oral fluid samples from three different subjects were spiked with 2.0 ng/mL of PEth standards, and the responses were compared with a methanol solution containing the same amount of analytes. Possible carry-over was tested by injection of 200 ng/mL of the analytes, followed by blank methanol.

The stability of PEth forms during sample storage was determined by spiking a blank oral fluid sample with two concentrations of PEth (1.0 ng/mL and 20 ng/mL). Two sets of samples were prepared and stored at 4 and −20 °C, respectively, and the stability was examined over 30 days (4 °C) and 90 days (−20 °C). In an additional experiment, 5 PEth positive patient samples were re-analyzed after storage at −20 °C for 6 months.

Clinical study

In the method application, oral fluid samples were collected from 35 alcohol-dependent patients undergoing alcohol detoxification at the Stockholm Centre for Dependency Disorders (Beroendecentrum). The blood alcohol concentration (BAC) at the time of oral fluid sampling was estimated from a breathalyzer test.

The oral fluid samples were immediately put in a fridge and transported to the Karolinska University Laboratory Huddinge on the same day. In the laboratory, the samples were stored frozen at −20 °C until taken for analysis. In 20 cases, a venous whole blood sample was collected at the same time and analyzed by the in-house routine method for PEth 16:0/18:1 [25], essentially as described before [26].

Results

Method validation

For all PEth forms, the LOD and LLOQ were ≤0.13 ng/mL and ≤0.45 ng/mL, respectively, in spiked oral fluid (Table 1). The final LLOQ were set to the lowest concentration level in the calibration curve. The acquired CV were less than 20% and the recoveries 99%–114% for all samples (n=10).

Table 1:

Method limit of detection (LOD) and lower limit of quantification (LLOQ) for 3 PEth forms acquired from spiked blank oral fluid (0.2 ng/mL).

PEth 16:0/16:0PEth 16:0/18:1PEth 16:0/18:2
LOD, ng/mL0.070.130.1
LLOQ, ng/mL0.250.430.33
 Accuracy, % (n=10)99114108
 CV, %101111

  1. The LOD and LLOQ were calculated from the acquired values and corresponding sample chromatograms based on signal-to-noise ratios of >3 and >10, respectively.

Method linearity was evaluated over a concentration range from the individual LLOQ, followed by 1.0, 5.0, 20.0, 100, and 200 ng/mL in spiked blank oral fluid collected from a healthy non-drinking volunteer. A linear correlation was established between the spiked amount and the area ratio of native analyte/IS. Coefficients of determination (r2) values ≥0.99 were obtained for all PEth forms.

The intra- and inter-day imprecision and accuracy over 5 days yielded a total CV of 10%–18% with an accuracy of 92%–108% at 0.4 ng/mL of each PEth form, and 6.8%–7.7% total CV with 99%–109% accuracy at 20 ng/mL (Table 2). The total recoveries in blank oral fluid samples spiked with 2.0 ng/mL of each PEth (n=3) ranged between 99%–114%.

Table 2:

Intra- and inter-day imprecision and accuracy for measurement of 3 PEth forms in oral fluid samples.

AnalyteConc.Measured mean conc.Within-run CVa, (%)Between-run CVa, (%)Total CVa, (%)Accuracy
PEth 16:0/16:00.400.375.88.910.692
PEth 16:0/18:10.400.4111.07.810.8101
PEth 16:0/18:20.400.4313.412.118.0108
PEth 16:0/16:020.020.55.75.17.7102
PEth 16:0/18:120.019.86.22.86.899
PEth 16:0/18:220.021.96.83.27.5109

  1. aAnalysis was performed with five replicates per run over 5 days. Conc., concentration.

The experiments evaluating a possible matrix effect showed only negligible ion suppression or enhancement for all PEth forms with <10% variation of results (data not shown). In the interference experiment, no false positives were observed among oral fluid samples collected from 20 healthy individual volunteers. No carry-over in the LC system (i.e. all responses were <LOD) was observed.

In the stability test, no marked changes in concentrations were observed when spiked oral fluid samples were stored at 4 or −20 °C (Table 3). Furthermore, when 5 PEth-positive patient samples were re-analyzed after 6 months storage at −20 °C, excellent agreement with the original values was achieved (e.g. 90%–101% for PEth 16:0/18:1).

Table 3:

Results of a long-term stability test at two different concentrations of each PEth form in oral fluid samples stored at two different temperatures.

Analyte1.0 ng/mL stored at 4 °C1.0 ng/mL stored at −20 °C
Day 7Day 30Day 7Day 30Day 90
PEth 16:0/16:00.920.681.000.801.04
PEth 16:0/18:10.960.801.080.920.96
PEth 16:0/18:20.880.761.080.960.96
20.0 ng/mL stored at 4 °C20.0 ng/mL stored at −20 °C
Day 7Day 30Day 7Day 30Day 90
PEth 16:0/16:017.015.419.916.216.8
PEth 16:0/18:118.418.220.818.117.5
PEth 16:0/18:217.917.721.317.817.5

Identification and quantification of PEths in clinical samples

In the clinical application of the method involving patients undergoing alcohol detoxification following recent heavy drinking, the highest PEth concentration in oral fluid was always found for PEth 16:0/18:1 (Table 4). The PEth forms were identified based on accurate retention time and MS/MS fragmentation pattern (i.e. fatty acids), compared with reference substances. The monitored ion transitions were selective for isobaric compounds containing the phosphatidylethanol moity. Out of the total of 35 samples, the number of samples containing PEth 16:0/16:0, PEth 16:0/18:2, and PEth 16:0/18:1 were 22 (63%), 30 (86%) and 34 (97%), respectively. In 22 samples where all 3 PEth forms were measurable, the relative abundance based on ion intensities were estimated 6%, 22% and 72% for PEth 16:0/16:0, PEth 16:0/18:2, and PEth 16:0/18:1, respectively, demonstrating that PEth 16:0/18:1 was the dominant form in oral fluid. The PEth 16:0/18:1 values in oral fluid showed a weak positive correlation with the corresponding values in whole blood samples (r=0.50, p=0.026, n=20) (Figure 2). Furthermore, the PEth 16:0/18:1 concentration in oral fluid was significantly higher (p<0.0001, n=35) in patients who tested positive for ethanol in blood (i.e. showing a positive breathalyzer test) at the time of sampling (Figure 3).

Table 4:

Estimated concentrations of PEth (ng/mL) from 35 intoxicated patient samples (28 men and seven women).

SubjectsEstimated blood alcohol concentrtion, %PEth in oral fluid, ng/mL
PEth 16:0/16:0PEth 16:0/18:1PEth 16:0/18:2
Total (n=35)Rangend–7.5nd–5.95nd–55.3nd–10.2
Mean1.20.688.462.32
Median1.00.243.090.98

  1. nd, not detected (<LOD); see Table 1.

Figure 2: Correlation between PEth 16:0/18:1 concentrations in oral fluid and whole blood collected at the same time (r=0.50, p=0.026, n=20).
Figure 2:

Correlation between PEth 16:0/18:1 concentrations in oral fluid and whole blood collected at the same time (r=0.50, p=0.026, n=20).

Figure 3: Box-Whisker plot showing the oral fluid concentration of PEth 16:0/18:1 in patients testing negative or positive for ethanol in blood (BAC; i.e. based on a breathalyzer test) at the time of sample collection.The PEth concentrations were significantly higher (p<0.0001, n=35) in the ethanol-positive patients.
Figure 3:

Box-Whisker plot showing the oral fluid concentration of PEth 16:0/18:1 in patients testing negative or positive for ethanol in blood (BAC; i.e. based on a breathalyzer test) at the time of sample collection.

The PEth concentrations were significantly higher (p<0.0001, n=35) in the ethanol-positive patients.

Discussion

An LC-MS/MS method for measurement of three selected PEth forms in oral fluid samples collected using the Quantisal device was developed and validated, and successfully applied for the analysis of PEth in samples collected from patients undergoing alcohol detoxification. The analysis of PEth in biological samples is challenging, due to the presence of isobaric PEth species. Also phospholipids may be interfering, because it has been estimated that PEth can make up at maximum 1%–2% of the phospholipid concentration in cell membranes [5]. Nevertheless, using a clear extract of oral fluid through Folch extraction, a narrow bore analytical column, and a sensitive LC-MS/MS instrument, increased the method performance and allowed reaching a low quantification limit well below that reported for PEth in blood [5], [11], [16], [17], [27], [28]. This was necessary because the PEth concentration in oral fluid was found to be considerably lower than in blood. An advantage compared with blood was that the oral fluid in Quantisal buffer samples could be stored at −20 °C for at least 3 months without any deteriorating effect, whereas blood samples require storage at −80 °C [16]. This is possibly due to the lack of phospholipase D in the oral fluid samples.

The 3 PEth forms were variably identified in a majority (63%) of the oral fluid samples collected from the 35 alcohol patients. In agreement with the distribution of PEth forms in human blood [1], [7], PEth 16:0/18:1 was also the dominant form in oral fluid. This suggests that PEth testing in oral fluid samples may be used as a noninvasive alcohol biomarker, following further characterization with respect to necessary issues such as the sensitivity and half-life on abstinence and comparison with established alcohol biomarkers. Alcohol biomarkers are important clinical tools, as they can provide objective information on chronic excessive or relapse drinking and confirm alcohol abstinence. Carbohydrate-deficient transferrin (CDT) in serum and PEth in whole blood are already established long-term blood biomarkers. An alternative specimen for alcohol biomarkers is urine, which is used for testing of ethyl glucuronide (EtG) and ethyl sulfate (EtS), which have a short detection window (a few days) and are therefore suitable to detect recent single intake [29]. EtG can also be determined in hair and then used as an alcohol biomarker with long detection window [30].

One reason for performing this study was the observation that PEth was detectable in breath samples (i.e. exhaled breath particles) collected from chronic heavy drinkers [20]. The relation of PEth forms in exhaled breath particles was, however, not in agreement with the distribution of the corresponding phosphatidylcholines in lung fluid [19]. However, because the PEth concentration in oral fluid is about 1000-fold higher than in breath, it can be calculated that contamination of a breath sample with a few microliters of oral fluid would be sufficient to explain the previous findings of low PEth concentrations in breath. However, oral fluid contamination of breath samples, albeit possible [31], would be expected to occur randomly, demanding further studies.

In conclusion, the present results suggested that oral fluid may become an alternative noninvasive specimen for analysis of PEth as an alcohol biomarker. Because of the rather poor agreement with the PEth values in blood, the relation of oral fluid PEth to amount and time-course of alcohol exposure should be a subject for future studies. In addition, the origin of PEth in oral fluid also needs to be determined. In blood, PEth is predominantly distributed in the cell membranes, whereas the extra-cellular concentration of PEth is unknown. However, preliminary data in our laboratory suggested that the PEth concentration plasma is considerably lower than in oral fluid, indicating that this could not be the origin of the PEth found in oral fluid. Anyway, the results are promising and suggest that measurement of PEth in oral fluid samples might be developed into a clinically useful noninvasive alcohol biomarker.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: The research was supported by a research grant from Stockholm County Council (No 20140397). The funders had no role on study design and content of the manuscript.

  3. Employment and leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: None including funding organization.

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Received: 2016-8-24
Accepted: 2016-10-31
Published Online: 2016-12-19
Published in Print: 2017-8-28

©2017 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Mass spectrometry or immunoassay: est modus in rebus
  4. Reviews
  5. The use of liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring of antibiotics in cancer patients
  6. Tackling serum folate test in European countries within the health technology assessment paradigm: request appropriateness, assays and health outcomes
  7. Genetics and Molecular Diagnostics
  8. Genetic diagnosis of α1-antitrypsin deficiency using DNA from buccal swab and serum samples
  9. General Clinical Chemistry and Laboratory Medicine
  10. Serum triglyceride measurements: the commutability of reference materials and the accuracy of results
  11. Variant peptide detection utilizing mass spectrometry: laying the foundations for proteogenomic identification and validation
  12. Evaluation of two fully automated immunoassay based tests for the measurement of 1α,25-dihydroxyvitamin D in human serum and comparison with LC-MS/MS
  13. Parallel diurnal fluctuation of testosterone, androstenedione, dehydroepiandrosterone and 17OHprogesterone as assessed in serum and saliva: validation of a novel liquid chromatography-tandem mass spectrometry method for salivary steroid profiling
  14. Determination of oxycodone and its major metabolites noroxycodone and oxymorphone by ultra-high-performance liquid chromatography tandem mass spectrometry in plasma and urine: application to real cases
  15. Identification and quantitation of phosphatidylethanols in oral fluid by liquid chromatography-tandem mass spectrometry
  16. Relationship between plasma and salivary melatonin and cortisol investigated by LC-MS/MS
  17. Paramagnetic micro-particles as a tool for rapid quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma by UHPLC-MS/MS
  18. Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II)
  19. The serum concentrations of leptin and MCP-1 independently predict low back pain duration
  20. Immunoassay screening in urine for synthetic cannabinoids – an evaluation of the diagnostic efficiency
  21. Cancer Diagnostics
  22. Study of kallikrein-related peptidase 6 (KLK6) and its complex with α1-antitrypsin in biological fluids
  23. Cardiovascular Diseases
  24. A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide
  25. The natriuretic peptide MR-proANP predicts all-cause mortality and adverse outcome in community patients: a 10-year follow-up study
  26. CASZ1 loss-of-function mutation contributes to familial dilated cardiomyopathy
  27. Diabetes
  28. Evaluating new HbA1c methods for adoption by the IFCC and NGSP reference networks using international quality targets
  29. Infectious Diseases
  30. Analytical and diagnostic performance of two automated fecal calprotectin immunoassays for detection of inflammatory bowel disease
  31. Letters to the Editor
  32. Is fasting necessary for lipid profile determinations? Some considerations from the perspective of the clinical laboratory
  33. Precision of nonfasting lipid profiles should focus on clinical relevance rather than necessarily obtaining the least variation
  34. Triglyceride concentrations should be measured after elimination of free glycerol to exclude interindividual variations due to adiposity and fasting status
  35. Estimation of the reference interval for serum folate measured with assays traceable to the WHO International Standard
  36. Implausible elevation of peripheral thyroid hormones during therapy with a protein supplement
  37. Interference in Na+ measurements on the Siemens RAPIDPoint® 500 after nortriptyline intoxication: a case report
  38. Usefulness of maternal red cell antibodies to predict hemolytic disease of the fetus and newborn and significant neonatal hyperbilirubinemia: a retrospective study
  39. Improvement of the Sandell-Kolthoff reaction method (ammonium persulfate digestion) for the determination of iodine in urine samples
  40. Clinical use of targeted high-throughput whole-genome sequencing for a dengue virus variant
https://doi.org/10.1515/cclm-2016-0752
Keywords for this article
oral fluid; phosphatidylethanol (PEth) 16:0/18:1; quadrupole tandem mass spectrometer (LC-MS/MS)

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Mass spectrometry or immunoassay: est modus in rebus
  4. Reviews
  5. The use of liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring of antibiotics in cancer patients
  6. Tackling serum folate test in European countries within the health technology assessment paradigm: request appropriateness, assays and health outcomes
  7. Genetics and Molecular Diagnostics
  8. Genetic diagnosis of α1-antitrypsin deficiency using DNA from buccal swab and serum samples
  9. General Clinical Chemistry and Laboratory Medicine
  10. Serum triglyceride measurements: the commutability of reference materials and the accuracy of results
  11. Variant peptide detection utilizing mass spectrometry: laying the foundations for proteogenomic identification and validation
  12. Evaluation of two fully automated immunoassay based tests for the measurement of 1α,25-dihydroxyvitamin D in human serum and comparison with LC-MS/MS
  13. Parallel diurnal fluctuation of testosterone, androstenedione, dehydroepiandrosterone and 17OHprogesterone as assessed in serum and saliva: validation of a novel liquid chromatography-tandem mass spectrometry method for salivary steroid profiling
  14. Determination of oxycodone and its major metabolites noroxycodone and oxymorphone by ultra-high-performance liquid chromatography tandem mass spectrometry in plasma and urine: application to real cases
  15. Identification and quantitation of phosphatidylethanols in oral fluid by liquid chromatography-tandem mass spectrometry
  16. Relationship between plasma and salivary melatonin and cortisol investigated by LC-MS/MS
  17. Paramagnetic micro-particles as a tool for rapid quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma by UHPLC-MS/MS
  18. Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II)
  19. The serum concentrations of leptin and MCP-1 independently predict low back pain duration
  20. Immunoassay screening in urine for synthetic cannabinoids – an evaluation of the diagnostic efficiency
  21. Cancer Diagnostics
  22. Study of kallikrein-related peptidase 6 (KLK6) and its complex with α1-antitrypsin in biological fluids
  23. Cardiovascular Diseases
  24. A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide
  25. The natriuretic peptide MR-proANP predicts all-cause mortality and adverse outcome in community patients: a 10-year follow-up study
  26. CASZ1 loss-of-function mutation contributes to familial dilated cardiomyopathy
  27. Diabetes
  28. Evaluating new HbA1c methods for adoption by the IFCC and NGSP reference networks using international quality targets
  29. Infectious Diseases
  30. Analytical and diagnostic performance of two automated fecal calprotectin immunoassays for detection of inflammatory bowel disease
  31. Letters to the Editor
  32. Is fasting necessary for lipid profile determinations? Some considerations from the perspective of the clinical laboratory
  33. Precision of nonfasting lipid profiles should focus on clinical relevance rather than necessarily obtaining the least variation
  34. Triglyceride concentrations should be measured after elimination of free glycerol to exclude interindividual variations due to adiposity and fasting status
  35. Estimation of the reference interval for serum folate measured with assays traceable to the WHO International Standard
  36. Implausible elevation of peripheral thyroid hormones during therapy with a protein supplement
  37. Interference in Na+ measurements on the Siemens RAPIDPoint® 500 after nortriptyline intoxication: a case report
  38. Usefulness of maternal red cell antibodies to predict hemolytic disease of the fetus and newborn and significant neonatal hyperbilirubinemia: a retrospective study
  39. Improvement of the Sandell-Kolthoff reaction method (ammonium persulfate digestion) for the determination of iodine in urine samples
  40. Clinical use of targeted high-throughput whole-genome sequencing for a dengue virus variant
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