Development of quantitative assay for simultaneous measurement of purine metabolites and creatinine in biobanked urine by liquid chromatography-tandem mass spectrometry

Figures & data

Figure 1. Diagram of purine degradation pathway.

Table 1. Chromatographic conditions.

		Time (min)	Flow rate (ml/min)	%A	Column volumes
Elution	Isocratic	0	0.25	6.5	4.9
		10.2	0.25	6.5
	gradient	10.22	0.3	6.6	3.3
		15.9	0.3	43.1
Washing		16.4	0.3	50	2.0
		19.36	0.3	50
Re-equilibration		19.54	0.3	6.5	9.5
		21.27	0.3	6.5
		21.28	0.5	6.5
		29.04	0.5	6.5
		29.05	0.25	6.5
		31.00	0.25	6.5

Table 2. Specific mass spectrometric parameters and retention times (RT) for the analytes and internal standards (IS).

Analytes/ISs	RT (min)	Parent ion (m/z)	Daughter ion (m/z)	Cone voltage (V)	Collision energy (eV)
Hypoxanthine	9.03	134.88	91.94	30	15
^13C5-hypoxanthine	9.03	139.91	95.89	30	15
Xanthine	9.98	150.92	107.93	30	15
^13C,^15N2-xanthine	9.98	153.90	109.92	30	15
Creatinine	10.71	111.94	40.84	30	15
D3-Creatinine	10.82	114.95	41.85	30	15
Allantoin	14.80	156.97	113.94	16	12
^13C2,^15N4-allantoin	14.80	162.96	118.00	16	12
Uric acid	15.21	166.92	123.91	30	14
1,3-^15N2-uric acid	15.21	168.95	124.94	30	14

Table 3. General mass spectrometric parameters.

Source
Desolvation gas (l/h)	900
Capillary (kV)	0.6
Cone gas (l/h)	150
Cone (V)	30
Source temperature (°C)	150
Desolvation temperature (°C)	350
Collision gas flow (ml/min)	0.14
Nebulizer gas flow (bar)	7
Analyzer
Low mass resolution Q1	3.05
High mass resolution Q1	14.82
Ion energy Q1	0.6
Low mass resolution Q2	2.95
High mass resolution Q2	15.08
Ion energy Q2	0.8

Table 4. Concentration of labeled standards in the IS mixture and non-labeled standards in the calibrators’ mixture.

Labeled standards	Resulting concentration in the mixture (µM)
^13C2,^15N4-allantoin	46.9	IS mixture
^13C,^15N2-xanthine	16.7
^13C5-hypoxanthine	26.1
D3-creatinine	6049
1,3-^15N2-uric acid	1147
Non-labeled standards
Allantoin	188.2	Calibrators mixture
Xanthine	120.6
Hypoxanthine	78.7
Creatinine	19595
Uric acid	3227

Table 5. Composition of four times concentrated AU.

Components	g/100 ml
Urea	8
NaCl	4
K2SO4	0.68
MgSO4 × 7H2O	0.48
K2HPO4 × 3H2O	0.70
KH2PO4	0.56

Figure 2. Matrix effects. The results are quadruplicates of different dilutions of over-concentrated urine samples. Obtained concentrations were multiplied by the corresponding dilution factor and normalized to the average concentration of 20× diluted sample taken as 100% The dots and whiskers represent mean ± SD. Stars above the dots represent significant mean difference at p-levels: * < 0.05, ** < 0.001, *** < 0.0001.

Figure 3. Storage stability. The results are quadruplicates of urine aliquots stored at different conditions for 5.5 years. The dots and whiskers represent mean ± SD. Stars above the dots represent significant mean differences at p-levels: * < 0.05, ** < 0.0003, *** < 0.0001.

Figure 4. Typical LC–MS/MS chromatograms of four purines and creatinine in human urine. x-axis: retention time (min); y-axis: relative abundance (%).

Table 6. Concentration of purine metabolites and creatinine in 1st void morning urine by LC-MS/MS.

Concentrations	Mean (µM)	95% CI (µM)	Median (µM)	Std. deviation (µM)	Range (µM)
Women (n = 34)
Hypoxanthine	25.0	16.6–33.3	16	23.55	5–128
Xanthine	39.1	28.2–50.0	32	30.74	8–171
Creatinine	9522	7096–11948	7401	6841.7	2897–33856
Allantoin	92.9	71.3–114.4	83	60.75	24–298
Uric acid	2084	1622–2545	1778	1301.1	682–6135
Men (n = 21)
Hypoxanthine	36.8	23.5–50.2	24	29.30	3–104
Xanthine	34.0	25.8–42.2	34	18.09	6–74
Creatinine	14242	11319–17165	14821	6421.9	5857–27539
Allantoin	120.4	84.8–156.0	111	78.19	26–351
Uric acid	2816	2231–3401	2730	1285.6	760–5479

Table 7. Inter-day variation for QC samples.

	QC	Uric acid	Allantoin	Creatinine	Hypoxanthine	Xanthine
No. of days	Low	7	7	7	7	7
	High	8	8	8	8	8
Mean of quadruplicates’ means, (µM)	Low	1175	73	9061	19	22
	High	4268	136	19168	110	125
Std. dev of means	Low	15	6	400	0.2	0.2
	High	86	1	1185	2	3
% CV of means	Low	1.3	8.2	4.4	1.1	0.7
	High	2.0	0.8	6.2	1.5	2.1

Table 8. Intra-day variation, CV%.

Days	1	2	3	4	5	6	7	8	9
n	9	6	9	9	9	9	11	8	2
Hypoxanthine	0.18	0.16	0.27	0.20	0.15	0.18	0.17	0.23	0.27
Xanthine	0.23	0.18	0.21	0.21	0.20	0.19	0.17	0.28	0.23
Creatinine	0.51	0.62	0.43	0.54	0.36	0.34	0.35	0.42	0.24
Allantoin	0.74	0.78	0.91	1.07	0.76	0.81	0.85	0.78	0.39
Uric acid	0.45	0.48	0.50	0.54	0.63	0.58	0.45	0.42	0.62

Figure 5. Dynamics of changes in creatinine concentration in quality controls (QC) samples. Circles: high QC; squares: low QC; dashed line: linear regression of high QC; R² = 0.894, slope −0.454 mM/day ± 0.0639 SE; dotted line: linear regression of low QC, R² = 0.841, slope −0.17 mM/day ± 0.033 SE.

Table 9. Freeze-thaw stability.

	Freeze-thaw cycles
		1	3	6
Uric acid	% ± SD	100.00 ± 0.43	100.01 ± 0.51	99.96 ± 0.14
	p		0.7	0.6
Allantoin	% ± SD	100.0 ± 1.46	102.0 ± 1.40	103.2 ± 0.55
	p		0.04*	0.004*
Creatinine	% ± SD	100.0 ± 0.40	99.8 ± 0.41	98.9 ± 0.21
	p		0.3	0.001*
Hypoxanthine	% ± SD	100.0 ± 0.15	99.6 ± 0.22	98.8 ± 0.16
	p		0.01*	0.00001*
Xanthine	% ± SD	100.0 ± 0.21	99.4 ± 0.27	98.4 ± 0.13
	p		0.001*	0.000002*

*The mean difference from values of the first freeze-thaw cycle is assumed to be significant at the p-level 0.05.

Table 10. Descriptive statistics for creatinine and UA measured using reference and index assays.

Methods	Median	Ranges	IQR	Mean	SD	SE	Lower 95% CI	Upper 95% CI
Reference test for creatinine (enzymatic)	9.0	3.0–31.0	7.0–15.0	10.8	6.3	0.88	9.0	12.6
Index test for creatinine (LC-MS/MS)	7.2	2.0–28.0	5.2–12.2	9.1	5.7	0.80	7.5	10.7
Reference test for UA (enzymatic colorimetric)	1991	619–5698	1378–3076	2364	1403	206.9	1948	2781
Index test for UA (LC-MS/MS)	2038	676–6338	1456–3133	2438	1468	216.5	2002	2874

Creatinine (mM, n = 51), UA (µM, n = 46).

Figure 6. Passing–Bablok regression analysis of the two methods for UA (A), and creatinine (B). Index test: LC-MS/MS method; reference test: enzymatic colorimetric assay (A), and enzymatic assay (B); diamonds: UA samples (n = 46); squares: creatinine samples (n = 51); green line: regression line; red lines: 95%CI, black line: y = x. Spearman's correlation coefficients (A) 0.964, (p = 5.6582E-27), intercept median (A) −3.539 (95% CI: −28.560 to 24.247), slope (A) 1.057 (95% CI: 1.041–1.074). Spearman's correlation coefficients (B) 0.869 (p = 1.289E-16), intercept median (B) −0.620 (95% CI: −0.979 to −0.161), slope (B) 0.921 (95% CI: 0.886–0.964).

Figure 7. Bland–Altman plot for urinary UA (A), and creatinine (B). Index test: LC-MS/MS method; reference test: enzymatic colorimetric assay (A), and enzymatic assay (B); diamonds: UA samples (n = 46); squares: creatinine samples (n = 51); green line: average delta %; red lines: 1.96SD. The mean bias A: −3.5% (1.96 SD, −32.0 to 25.0%) for the enzymatic colorimetric assay minus LC-MS/MS method; B: 18.6% (1.96 SD, −24.43 to 61.63%) for the enzymatic assay minus LC-MS/MS method.

Table 11. Comparison of variation coefficients for UA and creatinine.

	Reference assay		LC-MC/MS method
	CVA declared by the manufacturer	CVA locally determined	inter-day variability CV%
Uric acid	1^a %	1.5^a %	1.3^b – 2^c %
Creatinine	2.1^e %	3^e %	4.4^f − 6.2^g %

CV_A: coefficient of analytical variation. Reference assays: uric acid – enzymatic colorimetric assay; creatinine – enzymatic assay.

a: determined for concentration 292 µM, b: determined for concentration 98 µM, c: determined for concentration 356 µM, e: determined for concentration 74 µM, f: determined for concentration 755 µM, g: determined for concentration 1597 µM.

Data availability statement

The data that support the findings of this study are available from University Hospital of North Norway (UNN) – but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are, however, available from the authors upon reasonable request and with permission of UNN and the Regional committee for medical and health research ethics.