Determination of diazepam in aquatic products by ultra performance liquid chromatography-tandem mass spectrometry with pass-through solid phase extraction

doi:10.3724/SP.J.1123.2019.11028

Abstract

Abstract:

A method was developed for the determination of diazepam in aquatic products by pass-through solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The analyte was extracted with acetonitrile directly and purified on a Prime HLB solid phase extraction column (60 mg/3 mL). The separation was performed on an Acquity UPLC BEH C₁₈ column (100 mm×2.1 mm, 1.7 μm)using methanol-0.1% (v/v) formic acid aqueous solution as the mobile phase in gradient elution mode. Qualitative analysis was performed in the multiple reaction monitoring (MRM) mode. The analyte was quantified by matrix-matched external standard curves. The results showed good linear relationship in the range of 0.1-10 ng/mL, and the correlation coefficient (r²) was greater than 0.99. The spiked recoveries of diazepam were 88.2%-101.1% at the spiked levels of 1.5, 3.0 and 15.0 μg/kg, and both the intra-and inter-day precisions were less than 10%. The developed method is simple, rapid and accurate, and it can meet the requirements for diazepam determination in aquatic product samples.

Key words: ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), pass-through solid phase extraction, diazepam, aquatic products

SU Shufang, SUN Lizhen, XUE Xia, GONG Pixue, WEI Lili, LI Xinling, ZHU Jianhua, LIU Yanming, ZHANG Feng. Determination of diazepam in aquatic products by ultra performance liquid chromatography-tandem mass spectrometry with pass-through solid phase extraction[J]. Chinese Journal of Chromatography, 2020, 38(7): 791-797.

Figures/Tables 11

Fig. 1 Chemical structure of diazepam

Table 1 Precursor ion, daughter ion and other MS parameters of diazepam

Analyte	Precursor ion (m/z)	Daughter ion (m/z)	Declustering potential/V	Collision energy/eV
*Quantitative ion.
Diazepam	285.2	154.0	100	37
		193.0^*	100	38

Fig. 2 Extracted ion current chromatograms of diazepam in the standard solution (1 ng/mL)

Table 2 Recoveries of diazepam using different extraction methods (n=6)

Pretreatment method	Recoveries/% (RSDs/%)
Pretreatment method	Yellow croaker	Carp	Crucian	Shrimp
Enzymolysis	82.1 (6.9)	79.5 (9.2)	80.6 (7.5)	92.5 (5.7)
Non enzymolysis	103.0 (5.4)	109.0 (4.3)	94.5 (8.3)	101.0 (2.9)

Fig. 3 Effect of different extraction solvents on the recoveries of diazepam Solvent 1: acetonitrile; solvent 2: acetonitrile (containing 1% (v/v) acetic acid); solvent 3: acetonitrile (containing 1% (v/v) ammonium hydroxide); solvent 4: ethyl acetate.

Table 3 Matrix effects of diazepam using different purification methods

Purification method	Matrix effects/%
Purification method	Yellow croaker	Carp	Crucian	Shrimp 1	Shrimp 2
Prime HLB	6.7	3.9	7.6	10.1	5.5
QuEChERS	11.8	13.5	9.9	16.3	15.0
Non purification	55.2	58.9	37.6	68.0	56.2

Fig. 4 Effect of purification columns with different specifications on the recoveries of diazepam

Fig. 5 Effect of volume fractions of acetonitrile on the recoveries of diazepam in sample loading solutions

Fig. 6 Effect of the nitrogen blowing on the recoveries of the different samples (n=3)

Table 4 Linear equations, correlation coefficients (r2), LODs and LOQs of diazepam

Matrix	Linear equation	r²	LOD/(μg/kg)	LOQ/(μg/kg)
Y: peak area; X: mass concentration, ng/mL.
Yellow	Y=1.826×10⁵X+1.463×10⁴	0.9992	0.5	1.5
croaker
Shrimp	Y=2.021×10⁵X+5.873×10³	0.9970	0.5	1.5

Table 5 Recoveries and precisions of diazepam in aquatic products (n=6)

Matrix	Spiked level/(μg/kg)	Average recovery/%	Intra-day RSD/%	Inter-day RSD/%
Yellow croaker	1.5	95.4	7.0	5.9
	3.0	93.5	4.8	3.3
	15.0	101.1	5.2	5.1
Shrimp	1.5	88.2	2.3	7.7
	3.0	95.6	3.8	5.2
	15.0	97.3	6.4	5.0

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