Establishment on a LC-MS/MS and its performance characteristic evaluation for the determination of serum 25-hydroxyvitamin D

doi:10.3969/j.issn.1673-8640.2015.05.003

Abstract

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

CLC Number:

Q565

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.

Figures/Tables 7

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

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

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

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

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

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

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

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

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

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

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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