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The determination of LCPs in human serum using Cleanert PPT protein precipitation plates by LC-MS/MS

Application Introduction

Lyso-Phosphatidylcholines(LPCs), a kind of phospholipid, has multiple physiological functions and it is closely related with diabetes, atherosclerosis, lipid abnormality and other metabolic diseases and cardiovascular diseases.

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Introduction

Lyso-Phosphatidylcholines(LPCs), a kind of phospholipid, has multiple physiological functions and it is closely related with diabetes, atherosclerosis, lipid abnormality and other metabolic diseases and cardiovascular diseases. There is a significant change of LPCs in liver metabolism, liver diseases and hepatotoxicity. In recent years, LPCs has been involved in the research of liver diseases and chemical hepatoxoxicity model.

This application note describes the analysis of five LPCs in human serum using Cleanert PPT protein precipitation plates with LC-MS/MS. With Cleanert PPT protein precipitation plate, centrifugation and supernatant transfer are eliminated, reducing the chance of sample loss and saving time. Extraction gained from Cleanert PPT protein precipitation plate can be introduced into LC-MS/MS. Excellent method linearity is proved for each analyze, together with good accuracy and precision performances.

Experimental

Materials and Reagents

Cleanert PPT protein precipitation plates, Cleanert M96 Positive  Pressure Device, Cleanert V96 N2 Evaporator, Cleanert 96-well Collection Plate, Durashell C18(L) (3.0 × 50 mm, 5 mm, 150Å) were provided from Bonna-Agela Technologies; Standard samples of  LPC 14:0, LPC 15:0, LPC 16:0, LPC 17:0, LPC 18:0 were purchased from Avanti; Reserpine (internal standard); Methanol; Formic acid; Water; Ammonium formate purchased from Honeywell;Normal saline(0.9%);

Sample Preparation

Due to the difficulty of obtaining substrates with no target compounds, human serum was replaced by normal saline in this methodological experiment. In order to maintain the biological integrity of LPCs in human blood, all the methanol used in experiment contain 0.1% 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT).

In the calibration curve and reproducibility test, normal saline was used as the substrate. Add 5 mL standard solution into 20 mL normal saline, vortex and wait for further preparation. In spiking human serum test, serum is substrate. Add 5 mL standard solution into 20 mL serum, vortex well and wait for further preparation.

Transfer 150 mL methanol (including 100 ppb reserpine) to the Cleanert PPT, then add the premixing sample, vortex, wait for 10 min and then collect the elute into a collector plate with Cleanert M96 positive pressure device.

Set the temperature of Cleanert V96 N2 Evaporator to 30 °C and dry the extraction by nitrogen.

Reconstitute with 1 mL 80% methanol in water, vortex well. The sample was ready for LC-MS/MS analysis.

Instrumentation

LC-MS/MS, API 4000+. SCIEX

Column: Durashell C18(L), 3.0 × 50 mm, 5 mm, 150 Å

Temperature: 30 °C

Flow rates: 800 mL/min

Injection: 5 mL

Mobile phase: A: 0.1% formic acid in water with 25 mM ammonium formate; B: 0.1% formic acid in methanol

Ion source: ESI+

Scan mode: MRM

Table 1 Gradient

Total   Time(min)

A

B

0

70

30

0.5

50

50

1

0

100

4

0

100

4.1

70

30

5

STOP

Table 2 MS Parameters

Compounds

tR/min

Q1

Q3

DP

CE

Reserpine

2

609.3

448.2

140

40

609.3

195*

120

45

C14:0

2.46

468.4

285.3

110

40

468.4

450.4*

120

27

C15:0

2.54

482.3

299.5

120

36

482.3

464.2*

120

28

C16:0

2.63

496.4

478.2*

124

16

C17:0

2.72

510.4

327.2

120

30

510.4

492.4*

120

20

C18:0

2.83

524.5

341.5

115

28

524.5

506.2*

127

18

Note:*is the quantitative ion

Results and Discussion

Calibration curve and method sensitivity

Calibration was preformed using 5 mL standard working solutions with 20 mL normal saline to obtain six concentration lever (Table 3, S1-S7). Figure 3 shows the sensitivity of method.

Table 3 Concentration lever and sensitivity


C14:0

C15:0

C16:0

C17:0

C18:0

S1/ppm

0.125

0.25

3.75

0.375

2.5

S2/ppm

0.25

0.5

7.5

0.75

5

S3/ppm

0.5

1

15

1.5

10

S4/ppm

1.25

2.5

37.5

3.75

25

S5/ppm

2.5

5

75

7.5

50

S6/ppm

3.75

7.5

112.5

11.25

75

S7/ppm

7.5

15

225

22.5

150

Calibration Equation

y=0.725x+0.00264

y=0.212x-0.0256

y=0.212x+0.268

y=0.366x+0.00653

y=0.31x+0.287

R2

0.9965

0.9913

0.9956

0.9938

0.9961

LOQ/ppm

0.027

0.12

0.32

0.059

0.072

Accuracy and Precision

Take normal saline as substrate, conduct two recovery experiments with two parallel samples (concentrations S3andS5). Table 4 shows the experimental results.

In order to inspect and study the accuracy and precision of this method, 8 parallel tests were conducted separately and sample concentrations were S3 and S5. S3 is corresponding to LQC and

S5 to HQC. Take normal saline as substrate. Table 5 shows the experimental results.

Table 4 Recoveries of spiking samples in saline


C14:0

C15:0

C16:0

C17:0

C18:0

Spiking samples in low   concentration/ppm

1.25

2.5

37.5

3.75

25

Spiking samples in high   concentration /ppm

3.75

7.5

113

11.3

75

Measured concentration of   low concentration samples-1/ppm

1.29

2.28

38.1

3.31

24.3

Measured concentration of   low concentration samples-2/ppm

1.44

2.85

43

4.09

28.1

Recoveries of low   concentration samples-1

103.2%

91.2%

101.6%

88.3%

97.2%

Recoveries of low   concentration samples-2

114.4%

114.0%

114.7%

109.1%

112.4%

Measured concentration of   high concentration samples-1/ppm

3.44

6.77

102

9.83

69.9

Measured concentration of   high concentration samples-2/ppm

3.35

6.53

103

9.94

69

Recoveries of high   concentration samples-1

91.7%

90.3%

90.3%

87.0%

93.2%

Recoveries of high   concentration samples-2

89.3%

87.1%

91.2%

88.0%

92.0%

Table 5 Accuracy and Precision of spiking samples in saline (n=8)


C14:0

C15:0

C16:0

C17:0

C18:0

Recoveries   of LQC

96.3%

99.9%

103.6%

96.6%

100.0%

CV

6.3%

6.4%

8.5%

7.1%

7.0%

Recoveries   of HQC

96.8%

99.1%

97.5%

93.8%

97.7%

CV

11.3%

12.1%

12.3%

12.0%

11.3%

It can be seen from Table 4 and Table 5 that this method can be adopted to dispose and detect

LPCs in normal saline. In order to further inspect its adaptability to serum samples, human serum

was used in another recovery experiment and the result can be seen in Table 6. Because of the

large concentration of LPCs in human serum, two human serum samples were detested to deduct

endogenous LPCs. The equation of recovery of spike sample is:

 

 

 

CA—measured concentration of spiked samples

CM—average concentration of human serum samples

CS—spiked concentration

Table 6 Method performance of spiking samples in human serum


C14:0

C15:0

C16:0

C17:0

C18:0

Spiking samples in low   concentration/ppm

1.25

2.5

37.5

3.75

25

Spiking samples in high   concentration /ppm

3.75

7.5

113

11.3

75

Spiking samples in human serum-1/ppm

0.827

2.46

107

2.92

47.3

Spiking samples in human serum-2/ppm

0.678

2.21

92

2.45

39.4

Measured concentration of   low concentration samples-1/ppm

1.97

4.5

140

6.24

72

Measured concentration of   low concentration samples-2/ppm

2.17

4.99

136

6.67

70

Recoveries of low   concentration samples-1

97.4%

86.6%

108.0%

94.8%

114.6%

Recoveries of low   concentration samples-2

113.4%

106.2%

97.3%

106.3%

106.6%

Measured concentration of   high concentration samples-1/ppm

4.8

9.46

196

13.8

113

Measured concentration of   high concentration samples-2/ppm

4.72

9.69

216

14.8

122

Recoveries of high   concentration samples-1

107.9%

95.0%

85.4%

98.4%

92.9%

Recoveries of high   concentration samples-2

105.3%

98.1%

103.1%

107.2%

104.9%

Figure 1 Chromatogram of standard working solution S2

Figure 2 Chromatogram of spiking sample in human serum

Conclusion

This experiment purified the LPCs from normal saline and human serum by protein precipitation and detect by LC-MS/MS. The result shows that this method is simple and stable, easy and fast to detect LPCs in human serum. It is suitable for clinical study.

Ordering Information

Products

Specification

Cat.No

Durashell C18(L)

3.0 × 50mm,5μm,150Å

DC950503-L

Cleanert   PPT

96-well   plates, 1 mL Square well

96CD1025-M

Cleanert®   V96 N2 Evaporator

Adapt   to 96-well plate

NV96-G-S

Cleanert®   M96 Positive Pressure Device

Adapt   to 96-well plate

SPE-M96

96-well   Collection Plate

2.0mL/well, adapt   to 96-well plate

96SP1036-Y

 

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