液相色谱-串联质谱法检测血清25-羟基维生素D的方法建立和性能评价

doi:10.3969/j.issn.1673-8640.2015.05.003

摘要/Abstract

摘要： 目的建立定量检测血清25-羟基维生素D[25(OH)D]的液相色谱-串联质谱(LC-MS/MS)方法,并对此方法进行基本分析性能验证。方法使用Waters^® Xevo^TM TQ MS ACQUITY UPLC^®液质联用仪(LC-MS/MS)同时定量检测血清中的25(OH)D2和25(OH)D3含量。参照美国食品和药品管理局(FDA)的生物分析方法验证导则标准对所建方法进行线性、检出限、精密度和准确性等基本性能的验证。结果 LC-MS/MS检测25(OH)D2和25(OH)D3的线性范围为6.25~500.00 nmol/L;25(OH)D2和25(OH)D3的定量检出限分别为2.50和1.25 nmol/L;批内、批间变异系数(CV)分别为<4%、<6%;回收率为93.26%~112.16%;维生素D国际室间质量评价计划(DEQAS)室间质评结果偏倚<10%。结论建立的LC-MS/MS基本性能符合评价标准,能够灵敏且准确的检测出血清中25(OH)D2和25(OH)D3 的浓度。

关键词: 25羟基维生素D, 液相串联质谱, 性能验证

Abstract: Objective To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitation determination of serum 25-hydroxyvitamin D [25(OH)D] and perform the general method performance verification. Methods The LC-MS/MS was used for the quantitation determination of 25(OH)D2 and 25(OH)D3 in serum by Waters^® Xevo^TM TQ MS ACQUITY UPLC^® mass spectrometry instrument. According to US Food and Drug Administration (FDA)'s guideline (Guidance for Industry Bioanalytical Method Validation), the general method performance verification was performed for linearity, determination limit, precision and accuracy. Results The linear ranges of 25(OH)D2 and 25(OH)D3 were 6.25-500.00 nmol/L. The LC-MS/MS had quantitation determination limits of 2.50 nmol/L for 25(OH)D2 and 1.25 nmol/L for 25(OH)D3. The within-run and between-run coefficients of variation (CV) of 25(OH)D2 and 25(OH)D3 were <4% and <6%, respectively. The recovery rates were 93.26%-112.16%. The result of the Vitamin D External Quality Assessment Scheme (DEQAS) interlaboratory quality assessment had a bias <10%. Conclusions The basic performance of LC-MS/MS meets the evaluation standards, and LC-MS/MS is sensitive and accurate for detecting the concentrations of 25(OH)D2 and 25(OH)D3 in serum.

Key words: 25-hydroxyvitamin D, Liquid chromatography-tandem mass spectrometry, Performance verification

中图分类号:

Q565

宋斌斌, 秦嘉倩, 彭颖斐, 张春燕, 吴炯, 王蓓丽, 郭玮, 潘柏申. 液相色谱-串联质谱法检测血清25-羟基维生素D的方法建立和性能评价[J]. 检验医学, 2015, 30(5): 416-420.

SONG Binbin, QIN Jiaqian, PENG Yingfei, ZHANG Chunyan, WU Jiong, WANG Beili, GUO Wei, PAN Baishen. Establishment on a LC-MS/MS and its performance characteristic evaluation for the determination of serum 25-hydroxyvitamin D[J]. Laboratory Medicine, 2015, 30(5): 416-420.

图/表 7

表1 质谱测定参数设置

物质	母离子>子离子 (m/z)	锥孔电压 (kV)	碰撞能量 (eV)
25(OH)D2	413.35>355.30	16.00	8.00
25(OH)D2-d3	416.35>358.30	16.00	8.00
25(OH)D3	401.35>159.10	16.00	28.00
25(OH)D3-d6	407.35>159.10	16.00	28.00

图1 LC-MS/MS检测血清25(OH)D2和25(OH)D3的色谱图和标准曲线

表2 LC-MS/MS检测血清25(OH)D2、25(OH)D3的线性评价 (nmol/L)

分析物	水平	理论值	测定值1	测定值2	$x ?$	s	CV (%)	偏倚(%)
25(OH)D2	1	500.00	484.50	515.50	500.00	21.92	4.40	0.00
	2	250.00	247.50	249.75	248.63	1.59	0.60	-0.60
	3	125.00	129.50	124.00	126.75	3.89	3.10	1.40
	4	62.50	61.75	68.00	64.88	4.42	6.80	3.80
	5	25.00	22.00	21.75	21.88	0.18	0.80	-12.50
	6	12.50	11.75	13.00	12.38	0.88	7.10	-1.00
	7	6.25	6.50	7.00	6.75	0.35	5.20	8.00
25(OH)D3	1	500.00	490.25	528.50	509.38	27.05	5.30	1.90
	2	250.00	241.25	243.00	242.13	1.24	0.50	-3.20
	3	125.00	126.00	122.00	124.00	2.83	2.30	-0.80
	4	62.50	60.00	62.75	61.38	1.94	3.20	-1.80
	5	25.00	26.00	25.50	25.75	0.35	1.40	3.00
	6	12.50	11.75	13.75	12.75	1.41	11.10	2.00
	7	6.25	6.25	8.00	7.13	1.24	17.40	14.00

表2 LC-MS/MS检测血清25(OH)D2、25(OH)D3的线性评价 (nmol/L)

分析物	水平	理论值	测定值1	测定值2	$x ?$	s	CV (%)	偏倚(%)
25(OH)D2	1	500.00	484.50	515.50	500.00	21.92	4.40	0.00
	2	250.00	247.50	249.75	248.63	1.59	0.60	-0.60
	3	125.00	129.50	124.00	126.75	3.89	3.10	1.40
	4	62.50	61.75	68.00	64.88	4.42	6.80	3.80
	5	25.00	22.00	21.75	21.88	0.18	0.80	-12.50
	6	12.50	11.75	13.00	12.38	0.88	7.10	-1.00
	7	6.25	6.50	7.00	6.75	0.35	5.20	8.00
25(OH)D3	1	500.00	490.25	528.50	509.38	27.05	5.30	1.90
	2	250.00	241.25	243.00	242.13	1.24	0.50	-3.20
	3	125.00	126.00	122.00	124.00	2.83	2.30	-0.80
	4	62.50	60.00	62.75	61.38	1.94	3.20	-1.80
	5	25.00	26.00	25.50	25.75	0.35	1.40	3.00
	6	12.50	11.75	13.75	12.75	1.41	11.10	2.00
	7	6.25	6.25	8.00	7.13	1.24	17.40	14.00

表3 LC-MS/MS的定量检出限评估结果 (nmol/L)

项目	期望值	实测值1	实测值2	实测值3	实测值4	实测值5	$x ?$	s	CV (%)	偏倚(%)	S/N
25(OH)D2	2.50	2.25	2.75	2.00	2.50	2.75	2.45	0.33	13.30	-2.00	743∶1
	1.25	1.00	1.50	2.00	1.00	0.75	1.25	0.50	40.00	0.00	494∶1
	0.63	1.00	1.00	0.50	0.75	0.50	0.75	0.25	33.33	20.00	325∶1
25(OH)D3	2.50	2.75	2.75	3.25	3.25	2.50	2.90	0.34	11.57	16.00	141∶1
	1.25	1.75	1.25	1.25	1.25	1.25	1.35	0.22	16.56	8.00	70∶1
	0.63	1.00	1.00	1.00	1.00	1.25	1.05	0.11	10.65	68.00	47∶1

表3 LC-MS/MS的定量检出限评估结果 (nmol/L)

项目	期望值	实测值1	实测值2	实测值3	实测值4	实测值5	$x ?$	s	CV (%)	偏倚(%)	S/N
25(OH)D2	2.50	2.25	2.75	2.00	2.50	2.75	2.45	0.33	13.30	-2.00	743∶1
	1.25	1.00	1.50	2.00	1.00	0.75	1.25	0.50	40.00	0.00	494∶1
	0.63	1.00	1.00	0.50	0.75	0.50	0.75	0.25	33.33	20.00	325∶1
25(OH)D3	2.50	2.75	2.75	3.25	3.25	2.50	2.90	0.34	11.57	16.00	141∶1
	1.25	1.75	1.25	1.25	1.25	1.25	1.35	0.22	16.56	8.00	70∶1
	0.63	1.00	1.00	1.00	1.00	1.25	1.05	0.11	10.65	68.00	47∶1

表4 LC-MS/MS的精密度评估结果 [总25(OH)D]

样本	批内			批间
样本	$x ?$ (nmol/L)	s(nmol/L)	CV(%)	$x ?$ (nmol/L)	s(nmol/L)	CV(%)
样本1	13.22	0.51	3.84	12.97	0.67	5.20
样本2	119.02	3.72	3.12	119.32	6.18	5.18
样本3	334.40	7.74	2.31	330.15	11.02	3.34

表4 LC-MS/MS的精密度评估结果 [总25(OH)D]

样本	批内			批间
样本	$x ?$ (nmol/L)	s(nmol/L)	CV(%)	$x ?$ (nmol/L)	s(nmol/L)	CV(%)
样本1	13.22	0.51	3.84	12.97	0.67	5.20
样本2	119.02	3.72	3.12	119.32	6.18	5.18
样本3	334.40	7.74	2.31	330.15	11.02	3.34

表5 回收实验[25(OH)D2+25(OH)D3] (nmol/L)

样本浓度	加入量		理论浓度	实测值1	实测值2	实测值3	实测 $x ?$	回收率(%)
样本浓度	25(OH)D2		理论浓度	实测值1	实测值2	实测值3	实测 $x ?$	25(OH)D3
31.1	2.84	8.52	42.44	41.50	37.25	40.00	39.58	93.26
	8.52	2.84	42.44	44.75	44.00	43.00	43.92	103.49
	5.68	17.04	53.80	54.00	51.25	53.50	52.92	98.36
	17.04	5.68	53.80	56.25	55.50	52.25	54.67	101.62
63.3	13.39	40.18	116.87	121.25	121.50	121.00	121.25	103.75
	40.18	13.39	116.87	126.75	123.25	130.25	126.75	108.45
	26.79	80.36	170.44	161.50	173.00	186.50	173.67	101.90
	80.36	26.79	170.44	197.50	193.75	182.25	191.17	112.16

表5 回收实验[25(OH)D2+25(OH)D3] (nmol/L)

样本浓度	加入量		理论浓度	实测值1	实测值2	实测值3	实测 $x ?$	回收率(%)
样本浓度	25(OH)D2		理论浓度	实测值1	实测值2	实测值3	实测 $x ?$	25(OH)D3
31.1	2.84	8.52	42.44	41.50	37.25	40.00	39.58	93.26
	8.52	2.84	42.44	44.75	44.00	43.00	43.92	103.49
	5.68	17.04	53.80	54.00	51.25	53.50	52.92	98.36
	17.04	5.68	53.80	56.25	55.50	52.25	54.67	101.62
63.3	13.39	40.18	116.87	121.25	121.50	121.00	121.25	103.75
	40.18	13.39	116.87	126.75	123.25	130.25	126.75	108.45
	26.79	80.36	170.44	161.50	173.00	186.50	173.67	101.90
	80.36	26.79	170.44	197.50	193.75	182.25	191.17	112.16

表6 LC-MS/MS检测DEQAS样本总25(OH)D的结果

样本编号	测定值1(nmol/L)	测定值2(nmol/L)	$x ?$ (nmol/L)	同方法组 $x ?$ (nmol/L)	偏倚(%)
436	73.70	78.90	76.30	81.10	-5.90
437	34.20	28.20	31.20	35.10	-11.20
438	60.10	62.90	61.50	58.40	5.30
439	44.00	43.60	43.80	43.10	1.70
440	47.40	50.60	49.00	50.60	-3.10

表6 LC-MS/MS检测DEQAS样本总25(OH)D的结果

样本编号	测定值1(nmol/L)	测定值2(nmol/L)	$x ?$ (nmol/L)	同方法组 $x ?$ (nmol/L)	偏倚(%)
436	73.70	78.90	76.30	81.10	-5.90
437	34.20	28.20	31.20	35.10	-11.20
438	60.10	62.90	61.50	58.40	5.30
439	44.00	43.60	43.80	43.10	1.70
440	47.40	50.60	49.00	50.60	-3.10

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