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Improved Sensitivity for the Selective Quantitation of LTB4 in Human Plasma and Sputum via UPLC-MS/MSChad D. Moore, Dan Li, Aihua Liu, Laixin Wang and Min Meng

Overview

Introduction

Purpose • Development of a high throughput method for the isolation

and accurate determination of Leukotriene B4 (LTB4) from human samples

Method• LTB4 extracted from human plasma/sputum samples by

Supported-Liquid Extraction in a 96-well format

• Extracts were analyzed via UPLC-MS/MS on an AB Sciex® QTrap® 6500

Result• Accurate determination of LTB4 from 20.0-2,000 pg/mL using

calibrators in surrogate matrix

• Excellent accuracy and precision for human plasma and sputum QCs

• No matrix effects observed between biological and surrogate matrices

• LTB4 levels successfully detected below 200 pg/mL in 7 different lots of human plasma samples and 9 different lots of human sputum samples

Method

Results and Discussion

Results and Discussion continued

Conclusion

References

• Previous methods for detection of LTB4 used manual liquid-liquid extraction, and quantitation by UHPLC-MS/MS with a LLOQ of 200 pg/mL. As endogenous levels of LTB4 are typically below 200 pg/mL, these methods have limited practicality. Using a high-throughput 96-well SLE extraction and UPLC-MS/MS, we were able to achieve a LLOQ of 20.0 pg/mL. This LLOQ is comparable to ELISA assays currently used to measure LTB4. However, where ELISA assays have potential for cross-reactivity with LTB4 isomers, our UPLC-MS/MS method completely resolved LTB4 from its isomers. Demonstrating the practical utility of our method, analysis of 10 individual human plasma and sputum samples confi rmed the ability of the method to successfully quantitate endogenous levels of LTB4.

TABLE 1. Precision and Accuracy of QC Samples

QC Samples (Matrix)Theoretical

Concentration (pg/mL)

Calculated Concentration

(pg/mL)Accuracy % CV

High (Human Plasma) 1600 1512 94.5 1.93

High (Human Sputum) 1600 1673 104 2.75

Medium (Human Plasma) 800 778 97.3 3.47

Medium (Human Sputum) 800 771 96.5 4.00

Low (10 mg/mL BSA) 60 59.6 99.4 2.21

TABLE 2. LTB4 levels 10 Individual Human Donors

Human Sample No.Concentration in Plasma (pg/mL)

Concentration in Sputum (pg/mL)

1 102 23.8

2 < LLOQ 23.1

3 < LLOQ 46.8

4 162 45.6

5 < LLOQ 104

6 133 84.6

7 60.5 143

8 45.9 76.6

9 36.2 < LLOQ

10 155 159

Sample PreparationMatrix: Human plasma (K2EDTA) and sputum for Medium and High QC preparation. 10 mg/mL Bovine Serum Albumin (BSA) in PBS for Low QC and calibration standard preparation

Calibration range: 20-2000 pg/mL

Aliquot size: 200 µL

Extraction: Isolute® (Biotage AB Corporation) SLE+ Fixed well plate with methyl tert-butyl ether.

LC-MS/MSMass Spec: AB Sciex® QTRAP® 6500

Source and ionization: ESI (Positive ion mode)

Source Temperature: 500°C

SRM transition: LTB4: 335.2 → 195 m/z

LTB4-d4: 339.2 → 197 m/z

LC Column: UPLC® OST-C18, 2x100mm, 1.7 µm

LC Flow Rate: 0.45mL/min

LC Mobile phase: A: Formic Acid in Water

B: Mix of Acetonitrile and Methanol

LC program: Gradient with column diversion

Run time: 5.0 minutes

Figure 1. Structure of LTB4 and its Endogenous Isomers

Figure 2. Representative Chromatograms using Various LC Columns

(a) Waters BEH Phenyl UPLC column, 2.1x50 mm, 1.7 µm

Figure 3. Representative Chromatograms of Calibration Standards and Control Blank

(a) LLOQ Standard (20 pg/mL)

Figure 4. Calibration Curve for LTB4 (20.0- 2,000 pg/mL) in Surrogate Matrix (10 mg/mL BSA)

Figure 6. Chromatogram of LTB4 from Human Plasma Sample. * Indicate LTB4 Isomers.

Figure 7. Chromatogram of LTB4 from Human Sputum Sample. * Indicate LTB4 Isomers.

Figure 5. Internal Standard Area Plot for Sample Analysis Run.

(b) Waters CSH Phenyl-Hexyl HPLC column, 2.1x50 mm, 3.5 µm

(b) Standards 2 (40 pg/mL)

(c) Standards 3 (100 pg/mL)

(d) Control Blank

(c) Waters OST C18 UPLC column, 2.1x100 mm, 1.7 µm

Leukotriene B4 (LTB4) is one of a family of eicosanoid infl ammatory mediators recognized in numerous disease states. For this reason, LTB4 is used as a pharmacodynamics biomarker and a therapeutic target for the treatment of various infl ammatory diseases. While there are commercially available ELISA methods which have LLOQ of 10- 200 pg/mL, these methods have the disadvantages of non-specifi city because ELISA antibodies have varying cross-reactivity with LTB4 isomers 12-epi-LTB4, 6-trans-LTB4 and 6-trans-12-epi-LTB4 (Figure 1). As such, a rapid, selective, and sensitive method for the measurement of LTB4 is of great utility. Previous UPLC-MS/MS methods only measured LTB4 down to 200 pg/mL in human plasma or sputum (W Jian 2013; W Lin 2013). However, as average LTB4 levels are below 200 pg/mL, previous methods had limited usefulness. Utilizing the improved sensitivity of the API 6500, our laboratory developed a high throughput extraction and UPLC-MS/MS method which allows for the accurate determination of LTB4 at lower levels than previously reported.

Method Development

• LTB4 is an endogenous analyte which is present in blank plasma and sputum. To quantify endogenous analytes in biological matrix, there are two common approaches. One approach is to use a modifi ed or surrogate matrix, such as charcoal stripped plasma, PBS buffer or BSA. Another approach is to use a surrogate analyte, such as a stable isotope-labeled analog. Each approach has its own pros and cons. For this presentation, we chose the surrogate matrix approach and used 10 mg/mL BSA in PBS for the preparation of standard calibrators.

• LC development was challenging. There are three known isomers of LTB4 (12-epi-LTB4, 6-trans-LTB4 and 6-trans-12-epi-LTB4) which possess the same molecular weight and cannot be resolved using a conventional 5 µm LC column (Figure 1). Various UHPLC columns were evaluated. The best column that yielded suffi cient resolution was UPLC® OST-C18 (Waters Technologies Corporation), 2x100mm, 1.7 µm (Figure 2).

• In the publications, either manual LLE extraction using MtBE (W Lin, 2013) or semi-automated LLE on a Tomtec® (W Jian, 2013) were used. For this method, an Isolute® SLE+ Fixed well plate was utilized. This approach was much faster than either manual LLE or the semi-automated Tomtec LLE.

Method Qualifi cation

• In the qualifi cation batch, duplicate standard calibrators were prepared in 10mg/mL BSA in PBS buffer. The fi nal concentrations were: 20, 40, 100, 300, 600, 1200, 1800 and 2000 pg/mL (Figure 3). Five pools of quality control samples were prepared as follows: Low QC at 60 pg/mL (10mg/mL BSA in PBS buffer), Medium QC at 800 pg/mL (plasma and sputum), High QC at 1600 pg/mL (plasma and sputum). QC samples were extracted at n=6.

• As shown in Figure 4, using 10 mg/mL BSA in PBS as a surrogate matrix, the calibration curve was linear over the range 20.0 – 2,000 pg/mL (r2 = 0.9973, weight 1/x2). The accuracy and precision for QCs in all matrices was excellent (single digit %CV and accuracy) (Table 1).

• Internal Standard responses were consistent with all matrices tested, demonstrating there are minimal matrix effects across different matrices and that 10 mg/mL BSA is a suitable surrogate matrix for the human plasma and sputum (Figure 5) assays.

Incurred sample analysis

• In this batch, 10 human plasma individual lots and 10 human sputum individual lots were extracted and quantifi ed against standard calibrators prepared in 10mg/mL BSA in PBS buffer.

• 7 of 10 individual human plasma lots had detectable LTB4 levels (Table 2). The 7 plasma lots had a mean SD of 99.2 ± 47.8 pg/mL with the highest sample containing 155 pg/mL of LTB4.

• 9 of 10 individual human sputum lots had detectable LTB4 levels (Table 2). The 9 sputum lots had a mean SD of 78.4 ± 49.2 pg/mL with the highest sample containing 159 pg/mL of LTB4.

• LTB4 was chromatically resolved from its isomers in both human plasma and sputum samples (Figures 6 and 7).

Note: Empty Circles Indicate Control Blanks

Lin W, et al. A highly sensitive and selective method for the determination of leukotriene B4 (LTB4) in ex vivo stimulated human plasma by ultra-fast liquid chromatography–tandem mass spectrometry. J Chromatogr B (2013); 925:54–62.

Lian W, et al. Quantitation of leukotriene B4 in human sputum as a biomarker using UPLC–MS/MS. J Chromatogr B (2013); 932: 59–65.