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RAB10004 A Rapid Clean-up Procedure for Monitoring the Biomarker of Dimethylformamide in Hemoglobin by LC-MS/MS

Application Introduction

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3-Methyl-5-Isopropylhydantoin (MVH), a metabolite of DMF was selected as the biomarker to monitor occupational exposure to DMF. A rapid sample preparation method to extract MVH from hemoglobin was established utilizing Cleanert  MAS-B 96-well Plate (25mg/well). 3-Methy-5-isobutylhydantion (MIH) was used as internal standard which had similar structure with MVH.

Table 1 Information of MIH and MVH

Compound

Name

CAS

Molecular   weight

Structure

MIH

3-Methy-5-isobutylhydantion

675854-31-6

170.10

     

MVH

3-Methyl-5-Isopropylhydantoin

74310-99-9

156.18

 

Experimental Details

Solution preparation

Diluted the stock solution of MVH and MIH with methanol/water(1:1,v/v) to required concentrations.

 

Sample Preparation

0.1g hemoglobin was weighed accurately and placed in 15mL tube. 0.25 mL of 20 μg/mL MIH and 4.75 mL of

hydrochloric acid/ acetic acid (1:1,v/v) was added into the tube and vortexed for 3 min. Then the tube was heated in

boiling water to degrade hemoglobin for 1 h. There might be bubbles emerged during the degradation process which was a spontaneous phenomenon. The tube was cooled to room temperature and brought the total volume of the sample to 5 mL by adding methanol/water (1:1,v/v). Then 200 μL of the treated sample was transferred into Cleanert® MAS-B 96-well Plate and mixed with 600 μL of 1% formic acid in acetonitrile. Finally, the treated sample was passed through the plate under vacuum for 2 min to 4 min. The fractions were collected and analyzed by LC-MS/MS.

 

Instrumentation

Instrumentation: LC-MS/MS, API 4000+

LC Instrument: Shimadzu LC-20A

HPLC Column: Venusil ASB C18, 150mm×2.1 mm, 5 μm, 100 Å

Mobile Phase: Acetonitrile/0.1 % formic acid-Water, 35:65(v/v)

Flow rate: 0.2 mL/min

Injection volume: 2 μL

Ion source: ESI Positive

Scan mode: MRM

Table 2 Precursor/Product Ions of MVH and MIH

Compounds

Retention   time (min)

Q1

Q3

DP

CE

MVH

2.8

157.2

129.1

61

16

71.9

66

23

55.2

65

37

MIH

3.4

171.1

143.1

62

18

86

66

22

69

62

29

 

Result

(1) Chromatogram

Table 3 LOD of MVH and MIH standard

Analyte

LOD

MVH

1.0ng/mL

MIH

1.0ng/mL

(2) Recoveries and Precision

Standard solutions of MVH and MIH were added in to hemoglobin samples accurately. The recoveries of the spiked

samples were listed in Table 4.

Figure 1 Chromatogram of MVH and MIH (250ng/mL)


Figure 2 Chromatogram of hemoglobin spiked with 50mg/kg MVH


Figure 3 Chromatogram of actual hemoglobin

 

Table 4 Recoveries and Precisions

Compound

Dosage(mg/kg)

Measurement(mg/kg)

Mean   recoveries (n=5)

RSD(n=5)

MVH

10

10.5

105.0%

4.6%

50

50.2

100.4%

3.5%

100

101.9

101.9%

2.9%

 

Cleanert MAS-B 96-well Plate is suitable to remove the matrix of hemoglobin which might interfere the analysis of MVH by LC-MS/MS. The usage of the 96-well plate is sufficient for high throughput sample clean-up procedure prior to LC-MS/MS. This experiment provided a solution for monitoring cumulative exposure to DMF.

 

Ordering Information

Products

Specification

Cat.No

Cleanert   MAS-B Plate

30mg/well

MS-B-0302W

Venusil  AQ C18

2.1   mm×150 mm, 5 μm, 100 Å

VA951502-0

96-well   collection plate

2.2   mL Squaral well

96SP2036-2

96-well   vacuum manifold

adapt   to 96well plate

VM96

Acetonitrile

HPLC,   4 L

AH015-4

1.5   mL vials

1.5   mL short thread vial, amber glass, label and filling lines

AV1111-0

1.5   mL vials caps

9   mm screw neck cap, center hole; red silicone/ white PTFE septa

AV2100-0

Filtration   membrane(Nylon)

φ13;   0.22 μm

AS021320-T

Disposable   Syringe

2   mL,needless

ZSQ-2ML



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