Research Article Special Focus Issue: Bioanalysis of Large Molecules by LC–MS

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Development and fit-for-purpose validation of a LC–MS/MS assay for fibrinogen peptide A quantitation in human plasma

Background: Fibrinopeptide A (FPA) is a plasma peptide, formed by the action of thrombin on fibrinogen during clog formation. FPA represents a direct indicator of thrombin activity and could potentially be used as a biomarker for anti-thrombotic therapy development. Results/Methodology: A LC-MS/MS assay with a high throughput solid phase extraction procedure was developed and validated to measure FPA in plasma. The lower limit-of-quantitation (LLOQ) of this assay was determined to be 0.16 nM. The inter- and intra-day%CV was 98% purity (high performance liquid chromatography [HPLC] and amino acid analysis analyzed), referred as FPA heavy in this study, was purchased from Sigma-Aldrich (MO, USA) as lyophilized solid. FPA heavy was dissolved in water with 0.1% formic acid. Stock solutions of FPA and FPA heavy was prepared in 400 μg/ml bovine serum albumin (BSA) and diluted further with 400 μg/ml BSA for assay use. Reagents

BSA was purchased from Thermo Scientific (IL, USA). Burdick & Jackson HPLC grade Water and Acetonitrile were purchased from Fisher Scientific (NJ, USA). HPLC grade methanol was purchased from Fisher Scientific (NJ, USA). Pierce Formic acid was purchased from Fisher Scientific (NJ, USA).

Bioanalysis (2014) 6(13), 1759–1766

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ISSN 1757-6180

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Research Article  Lin, Lassman, Weiner & Laterza

Key Terms Fibronopeptide A: Fibronopeptide A, a 16-amino-acid plasma peptide formed by cleavage of the fibrinogen α chain by thrombin during the conversion of fibrinogen to fibrin. Biomarker: Measured characteristic used as an indicator of normal biological process, disease process or pharmacologic response to therapeutic intervention. Biological variability: Natural variability of a biomarker due to physiologic different among subjects or within same subject over time.

Sample Collection

To identify the appropriate anticoagulant for FPA, a blood collection study was conducted to compare the variations of FPA collected in SCAT II tubes (Haematologic Technologies; VT, USA) and BD Vacutainer with sodium citrate. The concentrations of FPA found in sodium citrate tubes exhibited a less degree of tube-to-tube variation within the same subject. Therefore, sodium citrate plasma was used to determine FPA biological variability and the current validation study. Human plasma samples from 20 healthy volunteers were purchased from Bioreclamation (NY, USA). Blood was collected with sodium citrate as anticoagulant per vendor’s standard collection procedure. FPA assay

The FPA assay was consistent of two steps – solid phase extraction and LC–MS/MS analysis. FPA heavy was added to plasma as internal standard prior to SPE. A hydrophobic–hydrophilic interaction -based SPE procedure was optimized to extract FPA from plasma. SPE eluent was then directly injected to LC–MS for analysis. The concentration of FPA in plasma was measured with internal standard against a 11-point standard curve covering the range of endogenous FPA level. The assay performance was characterized by three levels of plasma QCs from healthy volunteers. Standard curve

Blank human plasma with no FPA can not be obtained, thus BSA solution was used as surrogate matrix to prepare FPA standard curve. With the application of stable isotope labeled FPA heavy as internal standard, the difference in matrix effect between plasma and BSA can be corrected. BAS was also used as carrier protein to minimize the loss of FPA during sample preparation due to nonspecific binding. The minimal concentration of BSA sufficient to prevent nonspecific binding was determined as 400 μg/ml. Stock solutions of 50 nM FPA in 400 μg/ml BSA and 25 nM FPA heavy in 400 μg/ml BSA were prepared sepa-

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Bioanalysis (2014) 6(13)

rately in single use aliquots and stored at -80ºC until use. A 11-point standard curve was prepared via 1:2 serial dilution from 50 nM FPA stock with 400 μg/ml BSA using a 350 μl Corning plate. After the standard curve solutions were prepared, 100 μl of each solution was transferred to a plate and 25 μl 25 nM FPA heavy was added to each well and mixed thoroughly. The standard curve solution was injected directly into LC–MS for analysis. Plasma sample extraction procedure

A Waters Oasis HLB 96-well μElution solid phase extract (SPE) plate packed with 30 μm particle size of polymeric reversed phase sorbent was used for plasma sample extraction. The SPE plate was pre-treated with a single wash of 200 μl methanol followed by two washes of 200 μl 0.1% formic acid in water. After thawing plasma samples at room temperature, samples were vortexed and 100 μl plasma was transferred to the SPE plate, followed by addition of 20 μl 25 nM FPA internal standard and 80 μl water with 0.1% formic acid. The plate was then placed on a Thermo Titer plate shaker to shake gently for 2 min for thorough mixing. The elution of SPE plate was performed on a Waters extraction plate manifold under vacuum with manual control through an air bleeding valve. The vacuum was controlled carefully to allow a flow rate of approximately one drop (40–50 μl) per second during the plasma loading step to achieve optimal binding between analyte and SPE sorbent. The SPE plate was subsequently washed two times with 200 μl of 0.1% formic acid in water. After wash, SPE plate was eluted with 100 μl elution buffer consisting of 0.1% formic acid in 25%/75% acetonitrile/water at one drop per second speed to a 96-well 2-ml deep well plate. The plate was then sealed with a rubber plate matt for subsequent LC–MS analysis. LC–MS Analysis

The analysis of SPE eluent was performed on a Waters (MA, USA) Acquity UPLC system coupled with an AB Sciex (Ontario, Canada) API 5000 mass spectrometer. A Waters Acquity UPLC BEH 2.1 × 50 mm, 1.7 μM, column in conjunction with a Waters Acquity BEH C18 Vanguard 2.1 × 5 mm guardcolumn was used. The analyses were performed with a flow rate of 0.6 ml/min, a column temperature of 50ºC and a binary mobile phase system consisting of A, water with 0.1% formic acid, and B, acetonitrile with 0.1% formic acid. A 7-min linear gradient was varied according the following program: 0 min (2% B), 0.2 min (2% B), 5 min (40% B), 5.1 min (85% B), 5.8 min (85% B) 5.81 min (2% B) and 7 min (2% B). The autosampler temperature was maintained at

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Development and fit-for-purpose validation of a LC–MS/MS assay for fibrinogen peptide A 

4ºC. For each sample, 40 μl was injected and the first 0.5 min of LC flow was diverted to waste to minimize source contamination. The API 5000 mass spectrom-

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eter was operated under MRM mode and positive electrospray ionization source was used with the ion spray voltage set at 5000 V and the heater temperature

A

2.50 450

Intensity (cps)

400 350

FPA heavy

300 250 200

1.96

150 100 1.78

50 0

1.5

1.6

1.7

1.90

1.8

1.9

2.63

2.02 2.0

2.1

2.2

2.3

2.4

2.5

2.6

2.99

2.80 2.7

2.8

2.9

3.37 3.04

3.0

3.1

3.2

3.3

3.4

Time (min)

Intensity (cps)

B

280 260 240 220 200 180 160 140 120 100 80 60 40 20 0

2.50

FPA in low QC 3.39

1.57 1.64 1.5

1.6

1.81 1.92 1.95 2.03 2.17 2.20 1.7

1.8

1.9

2.0

2.1

2.2

2.64 2.73

2.38 2.3

2.4

2.5

2.6

2.7

2.80 2.8

2.85 2.98 2.9

3.08

3.0

3.21

3.1

3.2

3.3

3.4

Time (min)

Intensity (cps)

C

2.50 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0

FPA in high QC

2.94 2.64 1.91

1.5

1.6

1.7

1.8

1.9

2.71

2.86

3.06

1.99 2.0

2.1

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.9

3.0

3.12 3.18

3.1

3.2

3.27

3.3

3.4

Time (min) Figure 1. Representative chromatograms.(A) FPA heavy spiked in plasma. (B) FPA in low QC plasma and (C) FPA in high QC plasma. 

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Research Article  Lin, Lassman, Weiner & Laterza set at 550 ºC. The operating parameters for gas and ion optics settings were: curtain gas 40, collision gas 7, gas one 50, gas two 50 and declustering potential 120 V. The resolution settings of Q1 and Q3 were set to unit mass. The multiple reaction monitoring (MRM) transition for FPA (MW 1536) was from the doublecharged precursor ion (m/z 769.3) to singly charged y11 ion (m/z 1077.8). The corresponding MRM transition for FPA heavy is from m/z 774.3 to m/z 1087.8. During assay development, no potential interference was found with the MRM transitions used for monitoring FPA and FPA heavy, respectively. As a result, no second MRM transition was incorporated in the assay procedure. The same collision energy (40 eV) and dwell time (100 ms) were used for FPA and FPA heavy. Analyst version 1.5 was used for both data acquisition and processing.

chromatograms of low QC and high QC are shown in  Figure 1 . QC samples were prepared in single-use tubes and stored at -80 ºC until use. Intra-assay precision was determined by measuring four replicates of QCs in one analytical run. Inter-assay precision was determined by measuring each QC sample over three runs conducted at different days. Analyte stability was assessed in plasma following up to three freeze–thaw cycles, as well as in extraction buffer stored in LC autosampler maintained at 4ºC up to 24 h. Dilution linearity was examined by measuring FPA concentration in three lots of plasma diluted with 400 μg/ml BSA up to 1:16. The SPE recovery was calculated by spiking FPA heavy into the three lots of plasma before and after SPE. The spike recovery was obtained from a low and a high concentration spike into three lots of plasma and a buffer solution (400 μg/ml BSA), respectively.

Analytical validation study design

The validation of FPA LC–MS/MS assay in this study followed a fit-for-purpose principle to establish a quantitative assay for measuring FPA in plasma. The following assay performance characteristics were assessed as part of analytical validation: sensitivity (defined by lower limit of quantification [LLOQ]), intra- and inter-assay precision, analyte stability, dilution linearity, SPE recovery, spike recover and biological variability. The biological variability of FPA was assessed in citrate-treated plasma (Bioreclamation, Inc.) from 20 healthy volunteers. Then, three lots of such plasma representing low (3.27 nM), medium (12.17 nM) and high (20.50 nM) levels of endogenous FPA were selected to prepare QCs without spiking any additional FPA standard. Representative

Results & discussion Sensitivity

Plasma with endogenous FPA was used to prepare QC in this assay. Therefore, LLOQ was not determined from QC prepared at LLOQ level. Instead, the LLOQ was estimated from five replicate measurements of independently prepared standard curves. For each concentration, the percent accuracy of measured concentration was calculated from individual curve. The mean and %CV of the same concentration from five curves was then determined (Table 1) . The LOQ was determined as the lowest standard concentration that can be accurately measured with %CV within 20%. The LOQ of this FPA assay was estimated to be 0.16 nM. The R 2 of the standard curve is above 0.99.

Table 1. LOQ determination from replicates (n = 5) of standard curve measurements. FPA standard (nM)

Calculated concentration (nM)

Mean (nM)

%CV

 

#1 

#2 

#3 

#4 

#5 

 

 

0.039

0.039

0.033

0.058

0.019

0.039

0.038

36.9

0.078

0.099

0.108

0.068

0.072

0.055

0.081

27.6

0.156

0.127

0.129

0.167

0.140

0.186

0.150

17.1

0.313

0.341

0.305

0.280

0.322

0.292

0.308

7.8

0.625

0.581

0.605

0.596

0.831

0.669

0.657

15.7

1.25

1.19

1.26

1.21

1.56

1.19

1.28

12.4

2.5

2.5

2.5

2.3

2.8

2.4

2.5

7.3

5

5

5

5

5

5

4.9

6.5

10

9

9

9

10

11

9.9

6.6

20

20

21

19

20

22

20.5

6.5

40

40

39

42

39

37

39.6

5.3

The limit of quantification was estimated to be 0.16 nM as it is the lowest standard concentration can be accurately measured with%CV less than 20%.

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Bioanalysis (2014) 6(13)

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Development and fit-for-purpose validation of a LC–MS/MS assay for fibrinogen peptide A 

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Table 2. Intra- and inter-assay precision.  

Intra-assay

Inter-assay

 

Low QC 

Medium QC 

High QC 

Low QC 

Medium QC 

High QC 

Mean

3.27

12.17

20.50

3.38

11.75

20.48

%CV

11.4

4.1

7.9

7.4

11.4

4.7

n

4

4

4

3

3

3

Table 3. Freeze–thaw stability for three plasma samples. Freeze–thaw cycles

FPA concentration (nM)

 

Plasma #1 

Plasma #2 

Plasma #3 

0

4.01

8.63

17.6

1

3.57 (89.0%)

9.22 (106.8%)

18.1 (102.3%)

2

3.94 (98.3%)

11.1 (128.6%)

18.0 (102.8%)

3

3.04 (75.8%)

10.5 (121.7%)

19.6 (111.4%)

FPA in cycle 1 to 3 as the percentage of FPA in cycle 0 was shown in parenthesis.

Precision

Recovery

Intra- and inter-assay precision was determined from 4.1 to 11.4% and from 4.7 to 11.4%, respectively (Table 2) . The precision of intra- and inter-assay was in the same range.

Total analyte recovery from SPE extraction was determined to range between 40–72%. This was determined by spiking labeled FPA to three different plasma specimens prior to extraction of the eluates of the same plasma specimens post-extraction. For typical spike recovery experiments, using labeled FPA for quantitation purposes, three different lots of plasma and a buffer (400 μg/ml BSA) were spiked with FPA at two concentrations (5 nM for low spike and 20 nM for high spike). The spike recovery was calculated as: % spike recovery = ([FPA concentration in spiked plasma – FPA concentration in no spiked plasma]/FPA concentration in buffer spike) x 100. The average recovery observed from the three lots of plasma tested was 102% (Table 6), which is within preciously established acceptable limits (80–120%).

Stability

The freeze thaw stability of FPA in plasma was examined on three lots of plasma subjected up to three freeze–thaw cycles. The measured FPA levels in plasma undergo one to three freeze–thaw cycles were found to be within the range of 75–130% of the FPA concentration in the original plasma (Table 3) . The stability of FPA level in SPE processed plasma samples in 4°C LC autosampler were examined by reinjecting a plate containing four sets of QC samples (low, medium and high) after 24 h. The measured FPA levels were found to within 83–120% of the original measured value. The average percentage of FPA levels after 24 h is 100.7% of the 0 h value and the%CV was 8.9% (Table 4) . This result suggested the loss of FPA in the extract plasma placed in the sample analysis plate due to none-specific binding was not significant.

Biological variability

Human plasma collected in sodium citrate tubes from 20 subjects was analyzed to determine the normal range of FPA concentration in healthy volunteers. Results are shown in Figure 2. The measured FPA ranged from 1 to 30 nM. The LOQ of this assay is ∼10 times lower than the lowest endogenous FPA found.

Dilution linearity

Dilution linearity was examined by measuring FPA concentration in three lots of plasma diluted with 400 μg/ml BSA up to 16-fold. A good linear correlation form each plasma lot was observed (R 2 > 0.995). The dilution-adjusted FPA concentrations were calculated for each sample. The adjusted FPA concentrations in the diluted plasma were found to be within 15% of the undiluted value (Table 5) .

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Conclusion A LC–MS/MS assay was developed and validated for the measurement of FPA in human plasma. The assay has good assay performance with intra- and inter-day precision less than 15%, sufficient sensitivity to measure endogenous FPA, dilution linearity up to 16-fold, freeze– thaw stability up to three cycles, good sample re-injection stability up to 24 h, and good spike recovery. The bio-

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Research Article  Lin, Lassman, Weiner & Laterza logical variability measure using this assay revealed that the FPA level in plasma sample from healthy volunteer was from 1 to 30 nM.

Future perspective The LC–MS/MS assay developed in this study would be applied to relevant clinical study to examine the

Table 4. Autosampler stability of solid phase extraction plasma sample over 24 h. Sample

FPA concentration (nM)

Percentage of FPA after 24 h to 0 h (%)

 

 

0 h 

24 h 

 

Low QC sample

#1

3.48

3.66

105.2

 

#2

3.49

3.50

100.3

 

#3

3.55

3.51

98.9

 

#4

3.01

3.08

102.3

Medium QC sample

#1

12.4

11.6

93.5

 

#2

10.0

12.0

120.0

 

#3

11.5

11.9

103.5

 

#4

13.1

10.9

83.2

High QC sample

#1

19.2

20.9

108.9

 

#2

20.8

20.9

100.5

 

#3

21.5

20.3

94.4

 

#4

20.4

19.8

97.1

Average change

 

 

 

100.7

%CV

 

 

 

8.9

Table 5. Plasma dilution linearity. Dilution factor

Corrected concentration (nM) (change from the undiluted)

 

Plasma #1 

Plasma #2 

Plasma #3 

Undiluted

22.30

12.50

17.50

1:2

20.2 (-9.4%)

11.16 (-10.7%)

18.34 (4.8%)

1:4

21.6 (-3.3%)

11.68 (-6.6%)

20.04 (14.5%)

1:8

21.68 (-2.8%)

11.76 (-5.9%)

19.6 (12.0%)

1:16

22.4 (0.4%)

12.11 (-3.1%)

20 (14.3%)

Table 6. Spike recovery of low and high concentration spike of FPA in plasma samples.  Matrix 

No spike (nM)

Spike (nM)

% Recovery

400 μg/ml BSA

0

4.02

 

Plasma #1

0.77

5.3

112.7

Plasma #2

13.8

17

79.6

Plasma #3

4.32

8.39

101.2

400 μg/ml BSA

0

15.8

 

Plasma #1

0.77

20.8

126.8

Plasma #2

13.8

30.2

103.8

Plasma #3

4.32

18.2

87.8

Low concentration spike

High concentration spike

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Bioanalysis (2014) 6(13)

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Development and fit-for-purpose validation of a LC–MS/MS assay for fibrinogen peptide A 

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35 30

FPA concentration (nM)

25

20 15

10 5

Plasma 710

Plasma 709

Plasma 708

Plasma 707

Plasma 706

Plasma 705

Plasma 704

Plasma 703

Plasma 702

Plasma 701

Plasma 700

Plasma 699

Plasma 698

Plasma 697

Plasma 696

Plasma 695

Plasma 694

Plasma 693

Plasma 692

Plasma 691

0

Plasma Figure 2. Measured fibronopeptide A in citrate plasma from 20 healthy volunteers. The endogenous FPA was found from 1 to 30 nM.

validity of using FPA as a viable clinical biomarker for anti-thrombotic therapy development.

No writing assistance was utilized in the production of this manuscript.

Financial & competing interests disclosure

Ethical conduct of research

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Executive summary • A LC–MS/MS assay was developed and validated for the measurement of fibronopeptide A (FPA) in human plasma. • The assay has good intra- and inter-day precision (%CV

MS assay for fibrinogen peptide A quantitation in human plasma.

Fibrinopeptide A (FPA) is a plasma peptide, formed by the action of thrombin on fibrinogen during clog formation. FPA represents a direct indicator of...
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