** C CORRESPONDING AUTHORORRESPONDING AUTHOR

Importance of Performing Incurred Sample Stability (ISS) for having a RuggedImportance of Performing Incurred Sample Stability (ISS) for having a Rugged and Accurate Omega-3 Bioanalytical Methodand Accurate Omega-3 Bioanalytical Method

Catherine Dicaire, Mathieu Lahaie, Jean-Nicholas Mess, Milton Furtado, Fabio Garofolo and Christopher Perkin*Catherine Dicaire, Mathieu Lahaie, Jean-Nicholas Mess, Milton Furtado, Fabio Garofolo and Christopher Perkin*Algorithme Pharma Inc., Laval (Montreal), QC, CanadaAlgorithme Pharma Inc., Laval (Montreal), QC, Canada

INTRODUCTIONINTRODUCTIONCONCLUSIONCONCLUSION

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential omega-3 fatty acids (Figure 1Figure 1). They are important for healthy cardiovascular, brain and retina functions. These supplements are offered mainly as ethyl esters (EE), synthetically produced forms of fats. The recent interest in having these compounds as prescribed drugs rather than over-the-counter (OTC) supplements is requiring the need to develop rugged and accurate quantitative bioanalytical assays. This case study shows an increase of EPA concentration over-time during stability testing of incurred samples. This phenomenon was not observed in QCs under the same conditions, thus explaining the need of performing ISS. Specific sample handling requirements were established in order to preserve sample integrity.

OVERVIEWOVERVIEW• PurposePurpose

– To investigate the short-term stability of free EPA in incurred samples.

– To show the necessity of performing incurred sample stability (ISS).

– To ensure a rugged and accurate bioanalytical assay.• MethodMethod

– An ISS evaluation was performed and the results triggered an investigation.

– The variation of endogenous levels of free EPA was monitored in plasma/blood generated from 12 healthy volunteers under various conditions.

– The stability of incurred samples was compared to the stability of quality control samples (QCs).

– A stabilization procedure was developed.• ResultsResults

– The endogenous level of EPA increased in incurred samples, while being stable in QCs.

– The increase was donor dependent.– This deconjugation mainly occurred during the

short-term stability at 4ºC.– The anti-coagulant used had an impact on conversion.– An acid preservative was needed to stabilize the incurred

samples.

METHODSMETHODS

RESULTSRESULTS

Figure 1: EPA and DHA StructuresFigure 1: EPA and DHA Structures

SAMPLE PREPARATIONSAMPLE PREPARATION• Calibration curves were prepared in proxy matrix and QCs

were prepared both in proxy and human plasma due to the presence of endogenous levels of EPA and DHA.

CHROMATOGRAPHYCHROMATOGRAPHY• Reverse-phase (C18) gradient elution using HPLC Agilent

Technologies Series 1100 pumps and autosampler

DETECTIONDETECTION• Turbo Ion Spray ESI(-) AB SCIEX LC-MS/MS API5000

The analytical method for EPA and DHA was fully validated including short-term, freeze-thaw and long-term stabilities of EPA and DHA in proxy and human plasma matrices (K2EDTA and K3EDTA) (partial results presented in Table 1Table 1).

At first, incurred samples reanalysis (ISR) was performed using two different aliquots for both analyses. This evaluation showed acceptable results. Subsequently, a second ISR was evaluated using the same aliquot for both analyses. Accordingly, this ISR measured both the method robustness and the stability of the samples since the second aliquot used had undergone minimally an extra freeze-thaw cycle and an extra short-term and long-term period; thus turning this ISR into an ISS. The results of this evaluation showed a positive trend for EPA, but not for DHA. To further investigate this phenomenon, the blood/plasma of 12 healthy volunteers was drawn and the stability of EPA and DHA endogenous levels were monitored.

Table 2: Investigation Results for EPA StabilityTable 2: Investigation Results for EPA Stability

Table 5: Stabilization Tests Results for EPA in human Table 5: Stabilization Tests Results for EPA in human plasmaplasma

Analyte SRM transitions (MRM)

EPA 301.2/257.2

EPA-D5 306.3/262.3

DHA 327.2/283.2

DHA-D5 332.3/288.3

Table 1: Stabilities of EPA and DHA in Human Plasma Table 1: Stabilities of EPA and DHA in Human Plasma (K(K22EDTA)EDTA)

Furthermore, the stability of incurred samples was compared with the stability of QC samples. Table 2 shows that the endogenous level of EPA increased steadily in incurred samples whereas it stayed constant in QC samples. It is suspected that the enzymatic activity of the freshly generated plasma was greater than that of the commercial plasma used for QC preparation. This difference could either be attributed to the age of the commercial plasma or the addition of organic solvent to the spiked samples.

The ISS evaluation clearly showed the need to stabilize the incurred samples before analysis. First, blood kinetics were performed in different anti-coagulants, using freshly drawn blood, to verify whole blood short-term stability. This evaluation showed that the samples were stable in blood for at least two hours, which gave enough time to generate the plasma (Table 3).

Table 3: Human Whole Blood (NaF/KOx vs.K3EDTA)Table 3: Human Whole Blood (NaF/KOx vs.K3EDTA) Kinetic Stability of EPA at a temperature of 4ºCKinetic Stability of EPA at a temperature of 4ºC

Plasma kinetics were then performed in different anti-coagulants in freshly generated plasma in order to verify the short-term stability of EPA before sample stabilization (Table 4). The results showed that a stabilization procedure needs to be applied to the incurred samples within six hours. Furthermore, the results showed a slight difference in stability for the different anti-coagulants used. The use of sodium fluoride/potassium oxalate seemed to slightly inhibit the conversion, which could be attributed to the NaF lipase and esterase inhibitor properties.

Table 4: Human Plasma (NaF/KOx vs.K3EDTA)Table 4: Human Plasma (NaF/KOx vs.K3EDTA) Kinetic Stability of EPA at a temperature of 4ºCKinetic Stability of EPA at a temperature of 4ºC

In order to further investigate the incurred sample stability of EPA in human plasma, different stabilization procedures were tried, i.e. addition of different enzyme inhibitors and acidification of plasma from different donors. The results of these tests are presented in Table 5 (one donor). The other donors showed similar results.

The 0.5% acidification procedure using NaF/KOx as anti-coagulant was chosen as optimal to prevent EPA deconjugation in incurred samples. The method was fully validated.

•The stability performed on incurred samples showed an increase in EPA concentration over-time.•The phenomenon was not observed in QC samples, probably because of different enzymatic activity between the fresh and the commercial plasma.•These results triggered an investigation which consisted of monitoring the endogenous levels of free EPA in freshly drawn blood/plasma.•The investigation demonstrated that an extended short-term storage of samples at 4ºC generated a considerable increase in free EPA.•The phenomenon was shown to be donor dependent.•The deconjugation was totally prevented in acidified plasma with NaF/KOx as an anti-coagulant. •The evaluation of the ISS along with a thorough investigation of the observed instability led to a better collection procedure and a more rugged and accurate method for the quantification of free EPA and DHA in human plasma.

Comp. Stab. Comp. Stab. Comp. Stab. Comp. Stab.

Concentration 37.9 33.4 781.8 727.7 125.0 110.3 2026.2 1861.6

% Nominal 100.2 88.2 103.2 96.0 98 86.5 104.7 96.2

% Deviation

Concentration 37.9 33.4 781.8 721 178.3 159.0 2154.0 2029.6

% Nominal 100.2 88.3 103.2 95.1 111.1 99.1 109.5 103.1

% Deviation

Concentration 52.5 52.1 688.3 746.3 312.4 303.7 2160.0 2044.8

% Nominal 99.6 98.6 91.8 99.5 100 97.2 101.4 96.0

% Deviation

DHA

Stability DurationLow QC High QC Low QC High QC

-5.8%

Short-Term Stability at a Temperature of 4ºC

26.4 Hours

-11.9% -6.9%

3 Cycles

-11.9% -7.8% -10.8%

-2.8% -5.3%

EPA

Long-Term Stability at a Temperature of -20ºC

47 Days

-0.7% 8.4%

-11.7% -8.1%

Freeze-Thaw Stability (Frozen at -20ºC and

Thawed at 4ºC)

The results of the investigation confirmed an increase in the concentrations for EPA. Furthermore, it showed that the increase was donor dependent. When three freeze-thaw cycles were performed on the incurred samples for a cumulative short-term of ~15 hours, the increase in concentrations varied from 15 – 95% for different donors. In addition, the freeze-thaw cycles did not have a significant impact on the conversion since different numbers of cycles (zero, one or three) with similar short-terms (approximately five hours) showed similar results. The major factor influencing the conversion was the short-term period, as a significant increase was observed for extended short-term even when the samples were left at 4ºC (5 hrs vs. 10 hrs vs. 15 hrs). (Table 2)

Freeze-Thaw Cycle: 0 0 3 1 2 3Cummulative Short-

Term (hours):0.5 hrs 5 hrs 5 hrs 5 hrs 10 hrs 15 hrs

29.1 34.0 35.5 35.5 37.7 40.816.9% 22.1% 21.9% 29.6% 40.3%

131.0 136.9 150.2 148.5 157.0 155.04.5% 14.6% 13.4% 19.8% 18.3%

115.0 123.1 132.6 131.5 138.5 143.07.0% 15.3% 14.3% 20.4% 24.3%

115.0 123.1 132.6 131.5 138.5 143.07.0% 15.3% 14.3% 20.4% 24.3%

28.0 37.2 39.0 39.7 46.0 54.333.1% 39.5% 41.9% 64.7% 94.4%

342.8 363.9 373.2 353.7 384.0 391.06.1% 8.9% 3.2% 12.0% 14.1%

34.6 38.6 41.7 42.7 47.2 52.311.5% 20.7% 23.5% 36.3% 51.3%

114.7 122.0 128.4 129.6 129.9 145.06.4% 12.0% 13.0% 13.3% 26.4%

56.5 60.9 62.6 65.6 68.3 76.77.8% 10.7% 16.0% 20.9% 35.7%

69.1 80.1 86.5 86.5 93.4 104.615.9% 25.2% 25.2% 35.2% 51.4%

55.8 59.2 64.2 66.2 68.1 71.26.1% 15.0% 18.7% 22.1% 27.6%

102.7 113.5 121.1 125.8 133.4 152.010.5% 17.8% 22.5% 29.9% 47.9%

40.7 N/AV N/AV 38.6 40.6 39.7-5.2% -0.2% -2.4%

756.4 N/AV N/AV 757.8 743.1 732.90.2% -1.8% -3.1%

QC7

Donor 04

Donor 09

Donor 10

Donor 07

Donor 08

Donor 05

Donor 06

(% deviation vs. 0 F/T-0.5 hrs ST)

Donor 11

Donor 12

QC5

Donor 03

Donor 01

Donor 02

Sample IDConcentration (ng/mL)

Kinetic Times (min)

Average Peak Area Ratio

(n=3)% Deviation

Average Peak Area Ratio

(n=3)% Deviation

0 0.0657 N/AP 0.0826 N/AP15 0.0645 -1.7% 0.0799 -3.3%30 0.0671 2.2% 0.0801 -3.1%60 0.0670 2.1% 0.0809 -2.2%90 0.0682 3.8% 0.0834 0.9%

120 0.0670 2.0% 0.0851 3.0%

NaF/KOx K3EDTA

A significant difference was observed between the stability of EPA in NaF/KOx anti-coagulant when compared to K2EDTA or K3EDTA, although the use of NaF/KOx did not completely prevent the conversion. The addition of extra enzyme inhibitors such as PMFS, Physostigmine, Halt™ or Complete™ did not further stabilize EPA. The acidification of the plasma (0.5%) showed a significant improvement for the residual conversion observed in NaF/KOx.

Concentration (ng/mL)

Concentration (ng/mL)

Concentration (ng/mL)

Concentration (ng/mL)

Fresh1 F/T Cycle +

~6 hr ST2 F/T Cycles +

~12 hr ST3 F/T Cycles +

~20 hr STN/AP -20ºC 140.2 194.2 38.5% 212.7 51.7% 233.3 66.4%N/AP -80ºC 175.9 25.5% 190.6 36.0% 198.0 41.2%P800 -80ºC 155.7 180.4 28.7% 191.7 36.8% 201.6 43.8%N/AP -20ºC 139.1 195.5 40.3% 208.8 49.9% 222.4 59.6%N/AP -80ºC 163.7 17.5% 173.1 24.3% 206.1 48.0%N/AP 103.0 116.7 13.3% 112.2 9.0% 119.5 16.0%PMFS 104.4 118.7 15.3% 121.5 18.0% 118.6 15.2%Physostigmine 107.4 119.0 15.5% 112.1 8.9% 134.4 30.6%0.05% acid 107.5 110.8 7.6% 115.9 12.6% 120.7 17.2%0.5% acid 104.0 105.2 2.2% 107.3 4.2% 108.4 5.3%Halt 106.2 111.9 8.6% 109.4 6.3% 114.9 11.6%Complete 106.8 108.0 4.9% 109.3 6.2% 115.6 12.3%

QC5 (40.9 ng/mL)

K3EDTA N/AP -80ºC 38.3 38.6 -5.7% 40.6 -0.7% 39.7 -2.9%

QC7 (756.4 ng/mL)

K3EDTA N/AP -80ºC 730.1 757.8 0.2% 743.1 -1.8% 732.9 -3.1%

DonorAnti-

coagulantPreservative

Storage temperature

% Dev. % Dev. % Dev.

-80ºC

03

K2EDTA

K3EDTA

NaF-KOx

Kinetic Times (h)

Average concentration (ng/mL) (n=3)

% DeviationAverage

concentration (ng/mL) (n=3)

% Deviation

0 38.2833 N/AP 47.1982 N/AP1.5 38.6452 0.9% 49.2925 4.4%3 40.0865 4.7% 50.5509 7.1%

4.5 41.3518 8.0% 52.5919 11.4%6 42.1229 10.0% 57.1200 21.0%

NaF/KOx K3EDTA