Abstract
Phosphatidylethanol (PEth) is a group of phospholipids detectable in red blood cells exclusively following ethanol consumption. The primary PEth analog, PEth 16:0/18:1, has an extended half-life in red cells, providing a long window of detection and tremendous potential for the quantification of cumulative alcohol consumption. We developed and validated an LC/MS-MS method to quantify PEth 16:0/18:1 in dried blood spots (DBS) for clinical research purposes. Method development and validation followed FDA guidance but expanded on prior published methods through the evaluation of additional DBS-specific factors such as sample hematocrit, punch location, and spot volume. This method was applied to the quantification of PEth in participant samples.
Keywords: Phosphatidylethanol, Alcohol, LC-MS/MS, Validation, Dried Blood Spots
1. Introduction1
In the presence of ethanol, phosphatidylcholine (PC) moieties in red blood cells (RBCs) undergo a transphosphatidylation reaction via Phospholipase D to form phosphatidylethanol (PEth) (Figure I) [1–4]. PEth exists as many different analogs; of the 48 identified, the most prevalent is PEth 16:0/18:1, which makes up approximately 37% of total PEth [5]. As this is the most prevalent analog of PEth, it is the one that most studies quantify and use for analyses.
Figure I.
Formation of phosphatidylethanol (PEth) in red blood cells from phosphatidylcholine (PC).
RBCs lack the enzyme needed for PEth degradation [6]; PEth therefore accumulates, with the 16:0/18:1 analog exhibiting a half-life of ~8 days [7] with a detection window of several weeks from last ingestion depending on the level of alcohol consumption. PEth also has excellent specificity and sensitivity relative to other alcohol biomarkers (~100%) [8–10].
There are several published methods to quantify PEth in whole blood [5, 11–16] as well as dried blood spots (DBS) [17–21]. DBS offer several advantages over whole blood-based methods including smaller sample volume, increased analyte stability, and ease of transportation, storage, and collection [22]. However, as DBS are a unique matrix, there are critical DBS-specific factors that must be validated to ensure accurate analyte quantification. For example, varying spot volumes and hematocrit levels may impact PEth quantification in DBS, and unless known, the analytical method may not account for these effects. Our laboratory (Colorado Antiviral Pharmacology Laboratory, CAVP) has long-standing experience developing DBS-based LC-MS/MS assays involving analytes within red blood cells, used for clinical research purposes [23–30], and have identified important factors to be considered for the quantification of samples collected in clinical trials. This manuscript assessed some of these important factors for PEth such as dilution integrity, freeze-thaw stability, punch stacking, and quantification in spot volumes <20 μL.
Our goal was to develop a robust method to quantify PEth 16:0/18:1 in DBS for clinical research purposes and perform a validation which followed FDA guidance [31] including assessment of crucial DBS-specific factors, defined by the Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology [32], which could impact PEth quantification in diverse research settings. By identifying variables that may impact PEth quantification in DBS, a more accurate interpretation of the result can be made. In addition, we employed the use of patient quality controls (PQCs) to provide a longitudinal assessment of inter-assay variability across runs to evaluate the consistency of the method, as well as participated in an external quality assessment (EQA) to compare the method’s quantification of PEth with that of other laboratories. The validated method was then applied to a clinical research study for the quantification of PEth in participant samples and subjected to incurred sample reanalysis (ISR) to illustrate the utility of the assay methodology.
2. Materials and Methods
2.1. Materials
PEth 16:0/18:1 was obtained from Avanti Polar Lipids (Alabaster, AL) as a sodium salt and Cerilliant (Round Rock, TX) as a 1 mg/mL solution. PEth 16:0/18:1-D5 internal standard (PEth-IS) was obtained from Echelon Biosciences Inc (Salt Lake City, UT). All solvents used were of HPLC grade or better and obtained from Fisher Scientific (Fairlawn, NJ). DBS were spotted onto Cytiva Whatman™ 903 Protein Saver Cards. A 7.0mm Accu-punch (Electron Microscopy Sciences; Hatfield, PA) was used to punch each DBS sample.
2.2. Preparation of DBS Standards and Quality Controls
Following informed consent, whole blood was obtained from individuals who do not consume alcohol and was screened to ensure undetectable PEth concentrations. This was used to create standards at PEth concentrations of 5 (lower limit of quantification, LLOQ), 10, 15, 25, 50, 100, 250, and 500 (upper limit of quantification, ULOQ) ng/mL, and quality controls (QCs) at PEth concentrations of 5 (QLLOQ), 12.5 (low, QL), 80 (medium, QM), and 400 (high, QH) ng/mL. 800, 1000, and 10,000 ng/mL dilution QCs were made for dilution integrity testing. All standards and QCs were prepared by adding 10 μL of an appropriately diluted 1 mg/mL PEth preparation stock in methanol (the spiking solution) to 990 μL of whole blood. Standard and QC spiking solutions were prepared from two separate 1 mL (1 mg/mL) PEth preparation stocks purchased from Cerilliant. Mixtures were left to equilibrate for one hour at ambient conditions and vortex mixed every 15 minutes. 25 μL of blood were used to spot each DBS onto Whatman™ 903 Protein Saver Cards, which were dried for three hours at room temperature before being placed in plastic DBS storage bags with a desiccant and stored at −80°C.
2.3. DBS Extraction
DBS were allowed to equilibrate to ambient conditions for 30 minutes prior to extraction. A central 7mm punch was taken from each DBS and placed in a 5 mL plastic cryovial. 2.0 mL of 50:50 acetonitrile:methanol (v:v) were then added, capped and vortex mixed briefly, and sonicated for 15 minutes. 1.6 mL of the supernatant and 50 μL of internal standard solution were added to a 16×100mm borosilicate glass tube, and the solution was vortex mixed briefly. Samples were dried under nitrogen at 40 °C for 22 minutes and reconstituted in 200 μL of 95:5 acetonitrile:methanol (v:v).
2.4. LC-MS/MS Conditions
The LC-MS/MS system consisted of a Vanquish UHPLC and Thermo Scientific TSQ Quantiva triple quadrupole mass spectrometer. A 2.1×100 mm Phenomenex Kinetex 2.6-μm Polar C18 100-Å analytical column was used for chromatographic separations. The isocratic mobile phase consisted of 70% of mobile phase A; MP A (70:30 acetonitrile:10 mM ammonium acetate (v:v) solution) and 30% of mobile phase B; MP B (100% 2-propanol). PEth and PEth-IS eluted at approximately 4 minutes. A column wash was employed after each injection. The MS/MS source was operated in negative ion mode. Precursor/product ions were monitored for quantification, 701.6/281.2 for PEth and 706.6/281.2 for PEth-IS.
2.5. Use of Reference Material
Reference material purity was carefully considered. Luginbühl et al. found that the regioisomeric purity of PEth reference material significantly impacted the quantification of unknown samples [33]. Amongst Cerilliant, Avanti Polar Lipids, and Chiron reference material, Cerilliant’s was found to have the greatest regioisomeric purity that mimicked that of authentic PEth. As a result, DBS prepared from Cerilliant reference material was primarily used for validation. However, Avanti Polar Lipids reference material was used to assess long-term DBS and preparation and spiking solution stability, as this was the reference material used to create stored samples available for this testing.
2.6. Validation Tests
As a full validation was performed, acceptance criteria for all validation tests were established a priori with the use of a validation plan based on FDA guidance [31].
Inter-day and intra-day accuracy and precision were assessed on three separate days using six replicates of QLLOQ, QL, QM, and QH levels run against a fresh standard curve. Acceptance criteria for back-calculated working standards followed FDA guidelines and were as follows: ±20% for LLOQ accuracy within a run and ±15% for all other levels. Accuracy and precision validation runs were accepted based solely on calibration standard performance. Inter-assay and intra-assay accuracy and precision were evaluated with validation quality control analysis. The assay was deemed valid with validation quality control performance within ±20% for QLLOQ accuracy and precision, and ±15% for all other levels.
Dilution accuracy and precision were assessed with 2x and 8x dilutions on an 800 ng/mL dilution QC (by adding PEth-IS to 0.8 and 0.2 mL of supernatant instead of the method’s standard 1.6 mL), 4x and 16x dilutions on a 1000 ng/mL dilution QC (by adding PEth-IS to 0.4 and 0.1 mL of supernatant instead of 1.6 mL), and a 32x dilution on a 10,000 ng/mL dilution QC (by adding PEth-IS to 0.05 mL of supernatant instead of 1.6 mL).
Punch stacking accuracy and precision were assessed by extracting 1×3mm, 2×3mm, and 3×3mm punches from QH DBS and comparing their peak area ratios (PARs) to extracted 1×7mm punches in five replicates each. A correction factor was obtained between each punch combination. An accuracy and precision run was then conducted in which six 1×3mm punch replicates taken from QM and QH samples were extracted, quantified off a standard curve, and multiplied by the correction factor.
PQCs were prepared from PEth-positive whole blood donors and used to assess accuracy and precision using clinically obtained DBS samples with authentic PEth. Whole blood was obtained from three individuals with varying amounts of PEth and spotted to create a minimum of 80 PQC cards with 5 DBS each. Precision assessment for the PQC levels was performed analogous to that of the assay QCs in three PQC validation runs with n=6 replicates at each level. Furthermore, a nominal concentration for each PQC level was established over n=30 independent assay runs. This nominal concentration allowed for the assessment of intra-assay and inter-assay accuracy in the PQC validation runs. PQC levels are maintained in each assay run performed for the quantitation of subject samples, allowing for intra-assay accuracy assessment and inter-assay assessment longitudinally during assay maintenance with authentic PEth DBS samples. Accuracy acceptance criteria is ±20% from nominal for all PQC levels.
To assess for inter-lab reproducibility, an EQA was conducted by The Society of PEth Research with laboratories quantifying PEth 16:0/18:1 in four authentic samples. Following the quantification of PEth in the samples sent to our lab, data were back sent to The Society of PEth Research, who used data from the 16 participating labs to define the target concentration of each sample as: median quantified concentration ± (2 × Horwitz SD). Z-scores were calculated for each measurement to evaluate the deviation from the target concentration, with an absolute z-score ≤2 as acceptable.
Matrix effects were assessed at QL, QM, and QH levels using six lots of extracted (Set 3), post-extracted (Set 2), and neat (Set 1) samples prepared at each QC level. Additionally, matrix effects slopes were generated for each of the lots using Set 2 samples to assess if ME had an effect on PEth quantification that exceeded acceptance criteria [34], defined as a slope CV of >5%.
Recovery was assessed by extracting PEth from DBS and then performing two subsequent extractions from the same spot to determine whether any additional PEth was extracted.
Specificity was assessed by extracting seven different lots of DBS obtained from individuals who do not consume alcohol. Any interfering peaks at the time of PEth’s retention must have been <20% the area count of the LLOQ.
PEth crosstalk was evaluated by extracting a ULOQ standard without IS and evaluating for a response in the IS channel. IS crosstalk in the respective parent PEth channel was evaluated by extraction of a Blank-IS sample. An LLOQ sample was extracted for comparison. Crosstalk was acceptable if the response in the ULOQ internal standard channel was <5% of the Blank-IS signal and if the response in the Blank-IS analyte channel was <20% of the LLOQ signal. PEth analog specificity was also investigated between PEth 16:0/18:2 and PEth 16:0/18:1 by injecting both analogs onto the system and looking for crosstalk between the two.
PEth carryover was evaluated by injecting an extracted ULOQ standard followed by a blank and was acceptable if it was <20% of the LLOQ signal and <5% of the internal standard signal.
For all PEth standard and QC stock solution preparation stability testing, comparison of the mean results of test and control samples should be within ±10%. For all whole blood and DBS stability testing, comparison of the mean results of test and control samples should be within ±15%. Within each stability test, all samples (control and test groups) were measured within the same run.
Room temperature stability of PEth preparation and spiking solutions in methanol was assessed by comparing the PARs of stock solutions kept at room temperature for 24 hours to the responses from equivalent concentration stock solutions stored at −20 °C. Long-term stability of PEth preparation and spiking solutions was assessed by comparing the PARs of solutions kept at −20 °C for 547 and 462 days, respectively, to freshly made solutions. Samples were prepared in triplicate.
The stability of PEth in EDTA whole blood was assessed by leaving a participant sample with authentic PEth at room temperature for 4 days. Spotting of the blood occurred at T=0 (1 hour), 2 hours, 4 hours, 8 hours, 24 hours, and 4 days after collection. Triplicates of spots obtained at each time point were extracted and compared to T=0.
Conditional stability of PEth in DBS was assessed with QL and QH samples extracted in triplicate and their mean concentration was compared to the nominal concentration, as well as to control QL and QH samples. Conditional stability included DBS undergoing three freeze-thaw cycles, room temperature for seven days, and long term stored at −80°C for 228 days.
Extracted sample stability was assessed by retaining a set of standards and six replicate QL and QH samples in the autosampler at 15 °C and reinjecting them after 15 days to assess standard curve and QC performance. Reinjection reproducibility was assessed by processing the QL and QH data off the originally injected standard curve.
Concomitant medication testing was performed by spiking extracted QL and QH samples with a solution of the following concomitant medications: abacavir, sofosbuvir and its metabolite GS-331007, ledipasvir, lamivudine, aspirin, oxycodone, acetaminophen, tenofovir alafenamide, emtricitabine, tenofovir, darunavir, elvitegravir, ritonavir, efavirenz, and cobicistat. Medications were chosen due to their relevance for INCLUD participants [35] and other populations under study by our group and collaborators. Additional control DBS were extracted, and concomitant medication data were deemed acceptable if the comparison of the mean concentration of test and control was within ±15%.
The effects of hematocrit, DBS spot volume, and punch location on PEth quantification using full (7mm) and partial (3mm) volumes were assessed. Hematocrit was assessed two ways. First, we used the hematocrit dilution approach whereby blank blood with a hematocrit of 45% was used to make aliquots of 35% and 55% hematocrit blood, as this range of hematocrit encompasses typical values (36% to 54%) [36]. Similar to our standard and QC preparation process, 990 mL of each aliquot was spiked with 10 uL of a Standard B spiking solution and spotted, to ensure that all samples would have the same final theoretical concentration. DBS were extracted in triplicate, and the PARs of each DBS hematocrit level were compared. Next, we tested the hematocrit effect by varying the hematocrits of patient samples with endogenous PEth. A participant sample containing authentic PEth with a hematocrit of 52% was obtained and plasma added to create additional aliquots of 32% and 42% hematocrit. Each of these aliquots were spotted as 10, 25, 50, and 75 uL spots. Triplicates of each spot type were extracted, including triplicate central and edge 3mm punches for the 25 uL spots, and triplicate central and edge 7mm punches for the 75 uL spots. The PARs of each combination were compared.
In the application of this method and to assess the assay’s reproducibility with participant samples, PEth was quantified in 384 DBS collected in the INCLUD study, a study of participants with Hepatitis C virus or Hepatitis C and HIV coinfection [35, 37]. ISR was conducted on 10% of samples randomly selected across the range of quantified concentrations, and acceptance criteria included at least 67% of the retested samples being quantified at concentrations within 20% of the average of the original and re-tested concentrations.
3. Results
3.1. Accuracy and Precision
A quadratic 1/x weighting was used for the standard curve as developmental data showed this best and most consistently fit the data. The mean percent deviation (accuracy) for all back-calculated standard concentrations used for accuracy and precision calibration curves were within ±7.1%. Precision (%CV) for all concentrations was ≤6.5%.
Inter-day and intra-day accuracy and precision of the QCs is shown in Table I. The inter-assay percent deviation was within ±9.4% for all QCs while precision was ≤9.0%. The intra-assay percent deviation was within ±14.3% for all QCs while precision was ≤12.8%. These data demonstrate that the assay meets assay validation criteria of precision and accuracy at all levels.
Table I.
Inter-day and intra-day PEth QC statistics.
| Inter-assay Statistics | Intra-assay Ranges | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Theoretical Concentration (ng/mL) |
5.00 | 12.5 | 80.0 | 400 | |||||
| Mean | 5.23 | 12.6 | 87.5 | 436 | LLOQ | Low QC |
Mid QC |
High QC |
|
| SD | 0.377 | 1.14 | 7.83 | 27.4 | %CV, min | 4.1 | 5.4 | 5.2 | 1.4 |
| CV (%) | 7.2 | 9.0 | 8.9 | 6.3 | %CV, max | 9.0 | 10.6 | 12.8 | 8.0 |
| Deviation from Nominal (%) | 4.6 | 0.7 | 9.4 | 9.1 | %error, min | 1.5 | −3.6 | 7.2 | 5.1 |
| n | 18 | 18 | 18 | 18 | %error, max | 6.6 | 7.4 | 12.5 | 14.3 |
Dilutions of 2x, 4x, 8x, 16x, and 32x were assessed with PEth QCs of 800, 1000, and 10,000 ng/mL. A minimum of 5 replicates were extracted per dilution. All dilution levels were shown to be accurate (within ±9.3%) and precise (≤8.8%) when multiplying uncorrected PEth values by the appropriate dilution factor to obtain corrected concentrations.
Partial volume accuracy and precision was assessed with a 1×3mm punch, first by validating the use of punch stacking, then by determining a correction factor between 1×3mm and 1×7mm punches. Punch stacking was shown to be accurate (within ±8.4%) and precise (≤8.0%) for 2×3mm and 3×3mm punches when compared to a 1×3mm punch. The determined correction factor for a 1×3mm punch to a 1×7mm punch was 4.6. The accuracy when applying this 1×3mm correction factor to the 2×3mm and 3×3mm punch stacked corrected result was within ±9.3%. Partial volume accuracy and precision results were within acceptance criteria with the correction factor applied. Accuracy was within ±5.2% and precision was ≤5.5%, showing that partial volume methods can be utilized with low volume spotted DBS samples if necessary.
The quantification of PEth in three separate PQC lots across 30 separate runs yielded the following nominal concentrations: a low concentration PQC (low PQC) of 23.9 ± 1.6 ng/mL (%CV: 6.7), a medium concentration PQC (medium PQC) of 44.8 ± 3.4 ng/mL (%CV: 7.6), and a high concentration PQC (high PQC) of 217 ± 16.4 ng/mL (%CV: 7.6). Inter-day and intra-day accuracy and precision of the PQCs is shown in Table II. The inter-assay percent deviation was within ±3.8% for all PQCs while precision was ≤7.2%. The intra-assay percent deviation was within ±9.4% for all PQCs while precision was ≤7.0%, demonstrating PQC precision and accuracy at all levels.
Table II.
Inter-day and intra-day PEth PQC statistics.
| Inter-assay Statistics | Intra-assay Ranges | ||||||
|---|---|---|---|---|---|---|---|
| Nominal Concentration (ng/mL) | 23.9 | 44.8 | 217 | ||||
| Mean | 24.4 | 45.7 | 225 | Low PQC |
Medium PQC |
High PQC |
|
| SD | 1.76 | 1.97 | 13.6 | %CV, min | 4.4 | 1.6 | 2.7 |
| CV (%) | 7.2 | 4.3 | 6.1 | %CV, max | 7.0 | 4.7 | 4.9 |
| Deviation from Nominal (%) | 2.3 | 2.0 | 3.8 | %error, min |
−3.5 | −0.8 | −1.8 |
| n | 18 | 18 | 18 | %error, max |
8.5 | 4.5 | 9.4 |
The monitored inter-assay variability of the PQC concentrations are shown in Figure II. These data demonstrate low inter-run variability in the extraction and quantification of authentic PEth in clinical samples using this assay.
Figure II.
Inter-assay variability of (a) low, (b) medium, and (c) high patient quality control (PQC) phosphatidylethanol (PEth) concentrations across n=54 runs. Each dot represents an individual extracted DBS. The solid line represents the established nominal concentration, while dashed lines represent concentrations ±20% that of the nominal concentration.
Target concentrations for the four samples quantified in the EQA were as follows according to The Society of PEth Research: 45 ± 11, 371 ± 67, 188 ± 38, and 17 ± 5 ng/mL. We quantified all samples within this range and without outliers, at 53, 395, 197, and 14 ng/mL, respectively. Our Z-scores for these samples were 0.85, 0.36, 0.24, and −0.63, respectively, demonstrating successful inter-lab comparisons of our quantification of PEth versus that of other laboratories.
3.2. Matrix Effects, Recovery, and Specificity/Selectivity
The mean matrix effect for PEth and PEth-IS were 71.0 ± 3.06% (%CV: 4.3) and 66.4 ± 2.15% (%CV: 3.2), respectively. The mean matrix effect for PAR data was 107 ± 2.15% (%CV: 2.0), showing that the analyte and IS exhibit the same matrix effect. The data are consistent with acceptable matrix effect differences between PEth and PEth-IS. Additionally, the slope precision (%CV) for ME slopes analysis was 1.6%.
Recovery was assessed after extraction of a Standard B DBS, where two subsequent extractions of the same spot only yielded an additional 5.7% and 1.6% PEth response compared to the initial response. These data indicate that the initial extraction yielded approximately 93% of the extractable PEth from DBS.
All seven DBS lots tested were free from any response that exceeded 20% of the LLOQ at the retention time of PEth and the PEth-IS. This demonstrates that other analytes and components in an individual’s blood will not interfere with PEth quantification.
3.3. Crosstalk/Carryover
Crosstalk evaluation showed no notable presence of PEth-IS in an extracted ULOQ standard, and no notable presence of PEth in an extracted Blank-IS sample. When PEth 16:0/18:2 was injected onto the system, no crosstalk was observed with PEth 16:0/18:1. In addition, monitoring 16:0/18:2 with its own product/precursors transitions of 699.5/279.2 showed baseline separation between where it eluted in comparison to 16:0/18:1. Carryover evaluation showed no notable presence of PEth or PEth-IS in a blank injected directly after a ULOQ standard.
3.4. Stability
Preparation of QL and QH spiking solutions prepared in methanol were found to be stable for at least 24 hours at room temperature (Table III). Preparation stock and QL and QH spiking solutions were also found to be stable at −20 °C for at least 547 and 462 days, respectively (Table III).
Table III.
Stability of PEth preparation stock and spiking Solutions prepared in methanol.
| Sample/Stability Condition |
CV (%) | Difference from Control (%) |
n | |
|---|---|---|---|---|
| Preparation Stock Solution | ||||
| Room Temperature1 | 3.9 | 1.1 | 3 | |
| Long-Term2 | 1.8 | −4.5 | 3 | |
| Spiking Solutions | ||||
| Room | QL | 0.7 | 2.7 | 3 |
| Temperature1 | QH | 5.3 | 0.6 | 3 |
| Long-Term3 | QL | 1.8 | 1.7 | 3 |
| QH | 0.5 | −2.6 | 3 | |
Kept at room temperature for 24 hours.
Stored at −20 C for 547 days.
Stored at −20 C for 462 days.
PEth was stable in whole blood at room temperature for at least 4 days before spotting (Table IV). PEth in DBS was stable at room temperature for at least 7 days, through at least three freeze-thaw cycles, and at −80 °C for at least 228 days (Table IV). PEth in extracted samples were stable in a 15 °C autosampler for at least 15 days, and when reinjected samples were quantified off the original curve, were stability-indicating (Table IV).
Table IV.
Stability of PEth in whole blood, dried blood spots, and extracted samples.
| Sample/Stability Condition | CV (%) | Deviation from Nominal (%) | Difference from Control (%) | n | |
|---|---|---|---|---|---|
| Whole Blood1 | 3.6 | NA | 2.2 | 3 | |
| Dried Blood Spots | |||||
| Room Temperature2 |
QL | 4.0 | 0.8 | 7.7 | 3 |
| QH | 7.0 | 1.8 | 9.4 | 3 | |
| Freeze-Thaw3 | QL | 5.9 | 2.7 | 1.4 | 3 |
| QH | 9.4 | 13.1 | 2.7 | 3 | |
| Long-Term4 | QL | 5.4 | 10.4 | 12.9 | 3 |
| QH | 2.3 | 7.3 | 9.4 | 3 | |
| Extracted Samples5 | |||||
| Autosampler | QL | 10.6 | 8.0 | 0.6 | 6 |
| QH | 2.0 | 5.5 | 0.4 | 6 | |
| Reinjection | QL | 11.3 | 0.9 | 7.7 | 6 |
| QH | 2.1 | 4.6 | 0.4 | 6 | |
Participant whole blood containing PEth was kept at room temperature for 4 days.
DBS were kept at room temperature for 7 days.
Three freeze-thaw cycles were evaluated with DBS kept at −80 C.
Long-term stability was evaluated at 228 days with DBS kept at −80 C.
Extracted samples were evaluated after 15 days of storage at 15 C.
3.5. Concomitant Medications
A cocktail of concomitant medications commonly used in persons with Hepatitis C and/or HIV were tested for their effect on PEth quantification (see 2.6 for complete list). These compounds had no notable effect on PEth quantification, with a ≤1.8% (%CV: 0.9) and ≤5.4% (%CV: 0.4) difference in QL and QH samples treated with the cocktail versus QL and QH control samples, respectively.
3.6. Additional DBS Testing
Differences in spot volumes and punch location had no notable effects on PEth quantification. However, changes in PEth quantification were seen with varying sample hematocrit. With the authentic PEth-positive blood, PEth concentrations were higher in higher hematocrit samples, with a 35.7% difference in PEth concentration observed between DBS samples of 32% and 52% hematocrit.
Using the blank blood used to create Standard B DBS of varying hematocrit, PEth concentrations were similarly higher in higher hematocrit samples, with a 28.1% difference in PEth concentration observed between DBS samples of 35% and 55% hematocrit.
3.7. Application
This assay was applied to the quantification of PEth in DBS collected from the INCLUD study. Briefly, INCLUD was a prospective, open-label study of 60 participants with active Hepatitis C infection and self-reported drug and/or alcohol use within 30 days of screening. Participants took ledipasvir/sofosbuvir (Harvoni®, Gilead, Foster City, CA) once daily for 12 weeks for Hepatitis C treatment. Self-reported drug and alcohol use, urine toxicology, and DBS for pharmacokinetic analyses were collected biweekly. PEth was quantified in 384 participant DBS. The range of PEth concentrations quantified was BLQ to 3656 ng/mL. 156 (41%) of samples were BLQ. Based on categorizations of PEth concentrations associated with different levels of drinking [38], there were: 179 (46.6%) instances of light or no consumption (<20 ng/mL of PEth), 97 (26.3%) instances of significant consumption (20–200 ng/mL of PEth), and 108 (28.1%) instances of heavy consumption (>200 ng/mL of PEth). 23 (38%) participants had all PEth concentrations in the light or no consumption range, and 13 (22%) participants had all PEth concentrations in the heavy consumption range.
ISR was conducted on 39 (10.2%) DBS across the range of quantified concentrations to assess the reproducibility of data across a range of concentrations. Concentrations on the ISR run were compared to the original run (Table V). 95% of samples met the 20% difference acceptance criteria, thus ISR passed within industry standards. The two samples that did not were either BLQ on the original run but quantifiable on the ISR run or vice versa. Although we classify these samples as failures, given that 20% variability is allowed around the LLOQ, it is possible to see these differences occur with samples of PEth concentrations on the LLOQ border. Original sample concentrations (log-transformed) plotted against their incurred percent differences showed no concentration-dependency in the reproducible quantification of PEth in patient samples (Figure III).
Table V.
Incurred sample reanalysis (ISR) of PEth quantification from the INCLUD study.
| ISR PEth Concentrations (ng/mL) | Original PEth Concentrations (ng/mL) | % Difference |
|---|---|---|
| BLQ | BLQ | 0.0 |
| BLQ | BLQ | 0.0 |
| BLQ | BLQ | 0.0 |
| BLQ | BLQ | 0.0 |
| 1642 | 1826 | −10.6 |
| 9.68 | 10.0 | −3.3 |
| 5.74 | BLQ | NA |
| BLQ | BLQ | 0.0 |
| 31.5 | 31.5 | 0.1 |
| 83.0 | 85.0 | −2.3 |
| 15.2 | 16.8 | −10.4 |
| 17.4 | 16.4 | 6.2 |
| 145 | 138 | 5.3 |
| 65.1 | 60.2 | 7.9 |
| 174 | 176 | −0.9 |
| 578 | 517 | 11.3 |
| 57.7 | 67.6 | −15.8 |
| 210 | 215 | −2.2 |
| 29.4 | 27.3 | 7.3 |
| 26.6 | 27.2 | −2.5 |
| 214 | 231 | −7.6 |
| 19.2 | 21.4 | −10.7 |
| 113 | 124 | −9.5 |
| 103 | 105 | −1.4 |
| 83.3 | 88.3 | −5.9 |
| 78.7 | 81.2 | −3.2 |
| 31.1 | 33.5 | −7.5 |
| 57.3 | 61.9 | −7.7 |
| BLQ | 5.41 | NA |
| 116 | 127 | −9.5 |
| 1376 | 1491 | −8.0 |
| 7.71 | 8.01 | −3.8 |
| 56.5 | 59.7 | −5.5 |
| 172 | 171 | 0.6 |
| 339 | 349 | −3.1 |
| 581 | 611 | −5.0 |
| 956 | 815 | 15.8 |
| 772 | 742 | 4.0 |
| 13.2 | 12.8 | 3.6 |
Figure III.
Incurred percent difference vs. original log-transformed PEth concentrations in samples used for INCLUD’s incurred sample reanalysis. Each data point represents a single sample. Samples with percent differences of 0% or NA are not included in this figure.
4. Discussion
The goal of this study was to develop and validate an LC-MS/MS assay to quantify PEth 16:0/18:1 in DBS. In addition to assessing standard validation parameters such as accuracy and precision, specificity, and matrix effects, DBS-specific factors that could affect PEth quantification were also assessed in order to interpret results more accurately. These additional testing data, such as spot volumes 10 to 75 μL, punch location, punch size (3mm and 7mm), varying hematocrit, etc., along with the assay’s LLOQ of 5 ng/mL, make this a robust assay that can accurately quantify PEth in a wide variety of clinical and research settings to capture a range of drinking practices [38].
The screening of whole blood used to create standards and QCs was found to be essential throughout the course of development, validation, and assay maintenance. Of 24 lots of whole blood purchased for our studies and requested to be obtained from individuals who do not consume alcohol, only six (25%) were blank. Of the rest, thirteen (54%) had concentrations between ≥5 and 100 ng/mL, two (8%) had concentrations between >100 and 1000 ng/mL, and three (13%) had concentrations >1000 ng/mL. Given this and the assay’s 5 ng/mL LLOQ, it was crucial to ensure that PEth was not present and that any possible signals from light alcohol consumption would not exceed 20% of the LLOQ response. Additionally, the use of PEth-IS is important as it is a stable labeled isotope of PEth and aids in minimizing any potential matrix effects that could occur in the ionization of the compounds during the electrospray process for the MS detection. The internal standard is typically added during the extraction step for most bioanalytical assay methods. However, this assay incorporates the use of internal standard prior to drying an aliquot of the extracted sample. Developmental studies showed this was the best method for utilizing the PEth-IS as the addition of PEth-IS to the DBS affected PEth quantification by ≥15%. We believe this is caused by the absorption of PEth-IS into the punched DBS paper during extraction. It was observed that PEth peak areas were precise, but PEth-IS peak areas were quite variable when the PEth-IS was added prior to extraction. The result was peak area ratios for quantification that were too variable for an accurate and precise method, while PEth peak area without PEth-IS was acceptable. Triplicates of Standard E DBS with the PEth-IS added during and post-extraction were utilized for this investigation, and it was observed that PEth-IS added pre-extraction yielded a 15.3% CV, while post-extraction yielded a 4.1% CV in PEth-IS variability. Adding PEth-IS post-extraction reduced IS variability to ≤5%, and PARs across runs were consistent. The use of PQCs with established inter- and intra-accuracy and precision across 54 independent runs, the successful determination of PEth in all authentic samples quantified as part of The Society of PEth Research’s EQA, as well as the application of this assay to 384 participant samples with a highly successful ISR, have shown that the method of IS addition and extraction provides a precise and accurate assay methodology. This will continue to be monitored through PQCs, with acceptance criteria within ±20% from their established nominal concentration.
Throughout validation, this assay was specific, accurate, and precise across a range of 5 to 500 ng/mL, with all intra-assay and inter-assay statistics within acceptance limits of 15%. Dilution accuracy and precision experiments showed that dilutions of 2x, 4x, 8x, 16x, and 32x were also accurate and precise. The quantification of PEth in low-volume spots was also validated via partial volume testing; as a result, spot volumes down to 10 μL can be quantified with the use of 3mm punches. This is essential testing as, in practice, volumetric sampling for DBS sampling is inconsistent across sites for large clinical trials. Varying spot volumes are often observed depending on the sample collection technique. As a result, whole-spot analysis is not a robust DBS methodology for clinical trial use or potentially application to home collection, thus leading to our validation of the PEth assay using 7mm and 3mm punches. This illustrates the utility of the assay for clinical research outcomes.
We showed PEth solutions in methanol and PEth in DBS to be stable in a variety of storage conditions. PEth standards and QCs in DBS were found to be stable for at least 9 months at −80 °C, which was seen by Bakhireva et. al, who also looked at the stability of PEth in DBS for 9 months [39]. We will continue to establish long-term stability for both spiked and authentic PEth in DBS. PQCs will be used to accomplish the latter, which will also continue to be used across all runs as assessments of inter-run extraction accuracy and variability.
The influences of crosstalk, carryover, matrix effects, and concomitant medications were found to not impact PEth quantification based on our established acceptance criteria. Punch location and spot volume also had no effect. However, there was a >15% impact on PEth quantification due to sample hematocrit. This is expected as PEth arises from RBC, and so the concentration of RBC will influence the assay result. As a result, we have modified the assay to utilize DBS standards and QCs made from blood with a hematocrit in the range of 42–45% to: 1) better represent the average hematocrit of the population, which will allow for the majority of the samples quantified to be essentially unaffected, and 2) lessen the effects of very low or very high sample hematocrit on PEth quantification using this mid-range hematocrit, which is an important consideration for clinical research.
There are limitations to this assay. Though we evaluated the effects of hematocrit on the PEth quantification of both DBS with spiked and authentic PEth, we did not assess the hematocrit’s effect on extraction recovery or matrix effects. However, the lots of blood used for matrix effects testing had hematocrits that ranged from 42% to 50%, and within this range, matrix effects were not observed. In addition, our assay did not utilize a qualifier ion, which has been used for some other DBS-based PEth assays [17–21]. Though a qualifier ion may be used when PEth is quantified for toxicologic or legal purposes, FDA and ICH guidelines do not require a qualifier ion and this assay was used for clinical research purposes. Our assay has demonstrated high accuracy and reproducibility without the use of a qualifier ion through data generated from the ISR, PQCs, and EQA.
By assessing additional DBS factors that could affect PEth’s quantification, we were able to adjust the method to ensure lesser variability in the data and more accurately interpret our results. By utilizing this validated assay to quantify PEth in participant samples and then reproduce those values through both a successful EQA, ISR, and the consistent quantification of PQCs across runs, we have generated data to support that the assay is robust, accurate, and precise in routine use. This fully validated LC-MS/MS assay for PEth in DBS has comprehensively assessed a variety of analytical factors and will have great utility in the quantification of PEth and its association with clinical outcomes.
Highlights.
Accurate quantification of phosphatidylethanol across spot volumes of 10 to 75 μL.
Stability data for reference solutions, dried blood spots, and extracted samples.
Adjustment of standard preparation to account for sample hematocrit effect.
Reproducible phosphatidylethanol quantification in over 400 authentic samples.
Successful external quality assessment illustrating inter-lab reproducibility.
Acknowledgements
We would like to thank Pang Xiong for generating PQC data and members of the Colorado Antiviral Pharmacology (CAVP) Laboratory.
Funding
This work was supported by the National Institute on Drug Abuse at the National Institutes of Health [R01 DA040499 to J.J.K.] and the National Institute on Alcohol Abuse and Alcoholism [R01 AA030483 to J.J.K., 1 F31 AA028977–01 to L.M.S].
Footnotes
DBS: dried blood spots, EQA: external quality assessment, ISR: incurred sample reanalysis, LLOQ: lower limit of quantification, PAR: peak area ratio, PC: phosphatidylcholine, PEth: phosphatidylethanol, PQC: patient quality control, QC: quality control, RBC: red blood cell, ULOQ: upper limit of quantification, USDTL: United States Drug Testing Laboratory
Declaration of interests
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Jennifer J Kiser reports financial support was provided by National Institutes of Health. Lana M Salah reports financial support was provided by National Institutes of Health.
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