Establishment and performance evaluation of candidate reference measurement procedure for the determination of immunosuppressive drugs in human whole blood by ID-LC-MS/MS

doi:10.3969/j.issn.1673-8640.2023.03.003

Abstract

Abstract:

Objective To establish a candidate reference measurement procedure for immunosuppressive drugs（tacrolimus，sirolimus，everolimus and cyclosporine A） in human whole blood by isotope-dilution liquid chromatography-tandem mass spectrometry（ID-LC-MS/MS），and to evaluate its analytical performance. Methods Human whole blood samples were prepared by a simple protein precipitation method，and ID-LC-MS/MS was used to determine tacrolimus，sirolimus，everolimus and cyclosporine A quantitatively. The performance，including linearity，limit of quantitation，relative matrix effect，precision and trueness，were evaluated. Results ID-LC-MS/MS allowed the determination of the 4 immunosuppressive drugs within a total runtime of 4.5 min. ID-LC-MS/MS allowed the determination of cyclosporine A in the range of 10-500 ng/mL. Tacrolimus，sirolimus and everolimus were analyzed in the range of 1-50 ng/mL. The limit of quantitation was 10 ng/mL for cyclosporine A and 1 ng/mL for the others. The relative matrix effects were ≤8.64%. The precisions for intra-day and inter-day coefficients of variation（CV） were ≤5%. The average recovery rates were found to be 98.84%-100.99% for the 4 immunosuppressive drugs. Expanded measurement uncertainties were found to be ≤7.91 % for tacrolimus（k=2），≤7.97 % for sirolimus（k=2），≤7.14 % for everolimus（k=2） and ≤7.91 % for cyclosporine A（k=2）. Conclusions The candidate reference measurement procedure for tacrolimus，sirolimus，everolimus and cyclosporine A in human whole blood by ID-LC-MS/MS has been established successfully，which can be used in value traceability and standardization.

Key words: Immunosuppressive drug, Isotope-dilution liquid chromatography-tandem mass spectrometry, Reference measurement procedure, Performance evaluation

CLC Number:

R446.1

YANG Xiaodong, LI Quanle, PAN Qingqing, ZHOU Jing, XIE Yangmin, ZOU Jihua, SHEN Min, ZHANG Man. Establishment and performance evaluation of candidate reference measurement procedure for the determination of immunosuppressive drugs in human whole blood by ID-LC-MS/MS[J]. Laboratory Medicine, 2023, 38(3): 215-222.

Figures/Tables 8

References 13

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名称	定量/定性	母离子/（m/z）	子离子/（m/z）	去簇电压/V	碰撞能量/V
他克莫司	定量	821.7	768.6	150	27
	定性	821.7	786.6	150	22
他克莫司-¹³Cd₄	定量	826.7	773.6	150	27
西罗莫司	定量	931.7	864.7	150	24
	定性	931.7	882.6	150	16
西罗莫司-d₃	定量	934.7	864.6	150	24
依维莫司	定量	975.7	908.7	150	24
	定性	975.6	926.7	150	18
依维莫司-d₄	定量	979.7	912.7	150	24
环孢素A	定量	1 219.9	1 202.9	150	50
	定性	1 219.9	1 184.9	150	47
环孢素A-d₄	定量	1 223.9	1 206.9	150	50

名称	定量/定性	母离子/（m/z）	子离子/（m/z）	去簇电压/V	碰撞能量/V
他克莫司	定量	821.7	768.6	150	27
	定性	821.7	786.6	150	22
他克莫司-¹³Cd₄	定量	826.7	773.6	150	27
西罗莫司	定量	931.7	864.7	150	24
	定性	931.7	882.6	150	16
西罗莫司-d₃	定量	934.7	864.6	150	24
依维莫司	定量	975.7	908.7	150	24
	定性	975.6	926.7	150	18
依维莫司-d₄	定量	979.7	912.7	150	24
环孢素A	定量	1 219.9	1 202.9	150	50
	定性	1 219.9	1 184.9	150	47
环孢素A-d₄	定量	1 223.9	1 206.9	150	50

项目	理论浓度/（ng/mL）	测定均值/（ng/mL）	CV/%	偏移/%
样本1
他克莫司	0.26	0.28	7.23	7.69
西罗莫司	0.26	0.25	7.92	-3.85
依维莫司	0.26	0.19	10.80	-26.92
环孢素A	2.54	0.91	13.14	-64.17
样本2
他克莫司	0.52	0.55	3.63	5.77
西罗莫司	0.52	0.53	7.57	1.92
依维莫司	0.52	0.47	4.27	-9.62
环孢素A	5.10	3.81	5.51	-25.29
样本3
他克莫司	0.78	0.83	3.60	6.41
西罗莫司	0.78	0.80	4.98	2.56
依维莫司	0.78	0.73	2.73	-6.41
环孢素A	7.62	6.56	3.97	-13.91
样本4
他克莫司	1.03	1.10	4.54	6.80
西罗莫司	1.02	1.07	3.74	4.90
依维莫司	1.03	1.02	3.93	-0.97
环孢素A	10.03	9.44	3.18	-5.88

项目	理论浓度/（ng/mL）	测定均值/（ng/mL）	CV/%	偏移/%
样本1
他克莫司	0.26	0.28	7.23	7.69
西罗莫司	0.26	0.25	7.92	-3.85
依维莫司	0.26	0.19	10.80	-26.92
环孢素A	2.54	0.91	13.14	-64.17
样本2
他克莫司	0.52	0.55	3.63	5.77
西罗莫司	0.52	0.53	7.57	1.92
依维莫司	0.52	0.47	4.27	-9.62
环孢素A	5.10	3.81	5.51	-25.29
样本3
他克莫司	0.78	0.83	3.60	6.41
西罗莫司	0.78	0.80	4.98	2.56
依维莫司	0.78	0.73	2.73	-6.41
环孢素A	7.62	6.56	3.97	-13.91
样本4
他克莫司	1.03	1.10	4.54	6.80
西罗莫司	1.02	1.07	3.74	4.90
依维莫司	1.03	1.02	3.93	-0.97
环孢素A	10.03	9.44	3.18	-5.88

项目	加入量/ng	测定值/ng	回收率/ %	平均回收率/%	CV/%	项目	加入量/ng	测定值/ng	回收率/ %	平均回收率/%	CV/%
他克莫司						依维莫司
加标样本1	0.493	0.484	98.17	99.63	1.54	加标样本1	0.495	0.491	99.19	100.99	1.12
	0.482	0.472	97.93				0.484	0.490	101.24
	0.486	0.489	100.62				0.488	0.499	102.25
	0.474	0.474	100.00				0.476	0.480	100.84
	0.487	0.494	101.44				0.489	0.496	101.43
加标样本2	2.435	2.391	98.19	99.04	1.09	加标样本2	2.444	2.477	101.35	99.40	1.66
	2.483	2.499	100.64				2.493	2.508	100.60
	2.459	2.407	97.89				2.468	2.458	99.60
	2.502	2.485	99.32				2.512	2.450	97.53
	2.451	2.431	99.18				2.460	2.409	97.93
加标样本3	4.988	4.916	98.56	99.27	0.60	加标样本3	5.007	5.013	100.12	100.76	0.63
	5.158	5.147	99.79				5.177	5.261	101.62
	4.848	4.821	99.44				4.866	4.874	100.16
	4.893	4.831	98.73				4.911	4.961	101.02
	4.826	4.819	99.85				4.844	4.887	100.89
西罗莫司						环孢素A
加标样本1	0.494	0.488	98.79	99.34	1.33	加标样本1	4.921	4.945	100.49	100.56	1.21
	0.483	0.487	100.83				4.815	4.830	100.31
	0.487	0.483	99.18				4.854	4.928	101.53
	0.476	0.466	97.90				4.737	4.822	101.79
	0.488	0.488	100.00				4.860	4.797	98.70
加标样本2	2.440	2.429	99.55	99.26	0.70	加标样本2	24.309	23.900	98.32	99.77	1.29
	2.489	2.482	99.72				24.795	25.168	101.50
	2.465	2.459	99.76				24.552	24.564	100.05
	2.508	2.487	99.16				24.982	25.058	100.30
	2.456	2.409	98.09				24.469	24.142	98.66
加标样本3	5.000	5.008	100.16	99.52	1.03	加标样本3	49.803	49.077	98.54	98.84	0.65
	5.170	5.071	98.09				51.495	50.972	98.98
	4.859	4.808	98.95				48.403	48.230	99.64
	4.904	4.889	99.69				48.851	48.413	99.10
	4.837	4.872	100.72				48.186	47.189	97.93