Comparison of Methods, Storage Conditions, and Time to Analysis of Serum and Urine Creatinine Measured from Microsamples by Liquid Chromatography Mass Spectrometery (LC/MS) vs. Jaffe

Abstract

Background

Measurement of serum creatinine (SCr) and urine creatinine (UCr) is regularly used in clinical and research settings. For small animal experiments and for studies in which sample collection is spare (i.e. neonatal cohorts), measuring SCr and UCr using tiny amounts of sample (as low as 10 mcl) would maximize exploration and minimize iatrogenic blood loss.

Methods

We performed an evaluation in six healthy adults to determine differences between SCr and UCr values in different methodologies and storage environments and time. Study was conducted using 20 mcl of sample. Analyses were done using two‐way repeated measures of ANOVA.

Results

Scr values showed no significant differences between LC/MS vs. Jaffe. However, the SCr using LC/MS method was lowest when measured immediately compared to other time points (F = 7.2; P< 0.001). Similarly, Jaffe measurements showed changes in the mean differences over time; however, these were not significant. UCr values were consistently higher using LC/MS than Jaffe (F = 19; P< 0.01), and UCr changed over time (F = 8.7; P < 0.02). In addition, the interaction term for method and time was also significant (F = 5.8; P < 0.04) which reflects the stability of the Jaffe measurements over time whereas the LC/MS measurements declined; especially after being frozen for 1 year (P < 0.001).

Conclusion

UCr measured by Jaffe is lower than samples measured by LC/MS. UCr measurements by LC/MS vary more over time, mostly due to the sample measured after 1 year; therefore, storage of urine for more than 90 days measured by LC/MS may provide altered results.

INTRODUCTION

Measurement of serum creatinine (SCr) is used to estimate kidney function, determine disease progression, and to screen for abrupt changes to diagnose acute kidney injury. Similarly, urine creatinine (UCr) is used in numerous applications in the practice and study of kidney disease including determination of tubular function (such as fractional excretion of sodium), determination of disease progression (for example urine protein/creatinine), and assessment of the accuracy of a timed urine collection. Urine biomarker evaluations commonly control for dilution/concentration.

With as little as 20 mcl of serum or urine, liquid chromatography tandem mass spectrometry (LC/MS) and Jaffe reaction can accurately measure SCr and UCr 1. In small animals, the volume of serum and urine available for measurement is sometimes sparse, necessitating use of very small volumes (as low as 10 mcl). In neonates, iatrogenic blood loss can be significant; thus, we have developed protocols to minimize blood loss and maximize the ability to perform clinical research. We have published data in neonates using microsamples of urine and blood to measure creatinine by both LC/MS and Jaffe methods 2, 3, 4, 5, 6, 7. Despite the accuracy of these tests, little is known about how these assays compare to one another after freeze/thaw cycles 8, in different storage conditions, and at extended periods of time.

In order to better understand how different techniques and sample storage practices affect the measurement of SCr and UCr using microsamples in humans, we performed an evaluation in six healthy adult volunteers. We tested differences between methodologies (LC/MS vs. Jaffe) under different storage environments (refrigerator vs. freezer) and across time (within 1 hr of sample collection, after 24, 1 mo, 3 mo, and 12 mo after collection) We tested the hypothesis that the urine and serum creatinine measurements would differ between 24‐hr refrigeration vs. freezing; Jaffe vs. LC/MS methods; and when frozen over time.

METHODS

Populations

After the Institutional Review Board (IRB) at the University of Alabama Birmingham granted approval for the study, six healthy nonpregnant, adult volunteers without known kidney/liver disease, who denied taking any medications known to interfere with creatinine determination, were recruited to participate in the study.

Sample Processing/Storage

Five milliliters of whole blood and 10 ml of urine were collected on one occasion for each subject. The samples were immediately centrifuged at 5000 rpm for 10 min. The supernatant of the serum and urine were batched individually in aliquots and then stored under either refrigerator or freezer (−80°C) and each of the aliquots were thawed individually and analyzed at several different time points, according to the following six conditions:

• Group A–measured within 2 hr of collection (reference).

• Group B–refrigerated shortly after centrifugation and measured 24 hr after collection.

• Group C–placed in −80°C freezer and measured 24 hr after collection.

• Group D–placed in −80°C freezer and measured 1 mo after collection.

• Group E–placed in −80°C freezer and measured 3 mo after collection.

• Group F–placed in −80°C freezer and measured 12 mo after collection.

Measurement of Urine and Serum Creatinine by LC‐MS/MS and the Jaffe Rate Method

Each serum and urine samples was measured using both LC/MS and Jaffe methods in batch at the stated time points. Briefly, LC/MS was performed using tandem mass spectrometry using multiple reaction monitoring and quantified via stable isotope dilution. Jaffe analysis was performed using a Beckman machine.

For LC‐MS/MS determinations, 10–20 μl of sample is deproteinated and diluted with stable isotope in a single step. Twenty microliters of diluted sample is subjected to isocratic HPLC with 10 mM ammonium acetate in 65% acetonitrile. Creatinine and d₃‐creatinine are detected by electrospray ionization tandem mass spectrometry transitions 114 > 44 and 117 > 47, respectively. Quantitation is achieved by comparing results to a standard curve 1. The following were used in all samples (MSMS Model: Quattro Micro API, Waters/Micromass, Milford, MA, USA); HPLC Model: Alliance 2795, Waters Corporation, Milford, MA, USA. HPLC column: TSK gelAmide‐80, Tosah Biosciences, King of Prussia, PA, USA. HPLC Model: Alliance 2795, Waters Corporation, Milford, MA, USA.

For the Jaffe method, 25 microliters of sample (in triplicate) is introduced to an automatic mixing/reading instrument (Creatinine Analyser II) containing picric acid solution. After a designated time, the creatinine‐alkaline picrate complex is measured by absorbance at 520 nm. The amount of creatinine in the sample is proportional to the absorbance of the complex as previously described 2.

Statistical Analysis

Data are expressed as mean ± SD. Analyses were done using two‐way repeated measures ANOVA (RM ANOVA). This provides three statistical tests—comparison of the two methods, comparison of the six time points, and an interaction term. The interaction term indicates if the two time courses are parallel. Comparisons over time points within method used one‐way RM ANOVA and the Neumann–Keuls procedure for determining where significant difference occurred. Any comparisons not listed are not statistically significant. Comparisons between two methods at one time point use paired t‐tests.

RESULTS

Table 1 shows the differences in SCr measurements over time for both the LC/MS and Jaffe methodologies and under different time/storage conditions. There are no significant differences between LC/MS vs. Jaffe SCr measurements. Likewise, there were no significant differences in SCr over time between groups. If we look at the two methods separately over time, we obtain the following results: in the LC/MS method, immediate measurement was lower than all others in all six subjects (F = 7.2; P < 0.001). Day 30 freezer was greater than day 1 freezer, day 1 fridge, and 1 year freezer. Similarly, Jaffe measurements showed changes in the mean differences over time; however, these were not statistically significant, likely due to higher variability and hence no differences were able to be shown.

Table 1.

Serum Creatinine Measurements by Time and Method

Sample	MS	JAFFE
Day 0	0.58 ± 0.11	0.67 ± 0.10
Day 1 fridge	0.62 ± 0.12	0.65 ± 0.19
Day 1 freezer	0.66 ± 0.16	0.68 ± 0.21
Day 30 freezer	0.69 ± 0.12	0.58 ± 0.15
Day 90 freezer	0.65 ± 0.13	0.67 ± 0.10
One year freezer	0.64 ± 0.12	0.70 ± 0.13
Two‐way RM ANOVA
Method effect	F = 0.2 P = 0.686
Time effect	F = 0.6 P = 0.540
Interaction	F = 2.0 P = 0.206

Table 2 shows the differences in UCr measurements over time for LC/MS vs. Jaffe methodologies across time/storage conditions. UCr was consistently higher using LC/MS than Jaffe (F = 19; P< 0.01) when explored as a group, and at each time point (differences between LC/MS and Jaffe at each time point all P < 0.001). Differences in UCr were detected over time (F = 8.7; P < 0.02). In addition, the interaction term for method and time was also significant (F = 5.8; P<0.04), which reflects the stability of the Jaffe measurements over time whereas the LC/MS values declined; especially after being frozen for 1 year. Follow‐up ANOVA highlights this as 1‐year freezer values measured by LC/MS are less than other values (P<0.001); whereas the 1‐year freezer samples were not statistically different vs. others measured by Jaffe (P = NS).

Table 2.

Urine Creatinine Measurements by Time and Method

Sample	MS	JAFFE
Day 0	90.6 ± 40.5	80.8 ± 36.3
Day 1 fridge	95.9 ± 47.5	78.0 ± 33.4
Day 1 freezer	94.0 ± 45.6	80.0 ± 36.1
Day 30 freezer	93.3 ± 41.9	78.7 ± 34.9
Day 90 freezer	92.9 ± 40.1	80.5 ± 35.8
One year freezer	75.7 ± 33.5	78.2 ± 38.6
Two‐way RM ANOVA
Method effect	F = 19 P < 0.01
Time effect	F = 8.7 P < 0.02
Interaction	F = 5.8 P < 0.04

DISCUSSION

In a small subset of healthy adults, we tested how different methods and storing conditions affected levels of SCr and UCr. We show that UCr values are significantly lower if measured by Jaffe vs. LC/MS methodology. The small differences seen are all less than any clinically meaningful differences; yet these differences need to be considered when assessing clinical and research and when comparing results determined by LC/MS vs. Jaffe. We did not, however, find any significant difference in SCr values between the different methods of measurement.

Over time, UCr measured by Jaffe remained stable over time and was consistently lower than LC/MS. UCr measurement using LC/MS appears to decline over time and there appeared to be a large drop in UCr values at the 1‐year measurement. Over time, SCr measurements of LC/MS and Jaffe were relatively stable over time. Changes over time did not differ between LS/MS and Jaffe method. We did not find differences in values stored in freezer vs. refrigerator at 24 hr.

Recent animal studies conducted by Schreier et al. measuring UCr using mass spectroscopy suggests that the effect of pH, osmolarity, and multiple freeze/thaw cycles had no effect on UCr creatinine values. However, they found similar findings to our study, whereby samples frozen at −20°C were stable for the first months, but changed when measured 12 months after collection.

We performed this study on a small number of healthy adults without kidney or liver disease and off any medications using two different methods to measure SCr and UCr in different storage conditions. We acknowledge several important limitations. A wider range of SCr values could affect our findings; thus, larger cohorts with samples obtained across different stages of chronic kidney disease, genders, ages, and clinical conditions need to be performed to be able to generalize our findings to a wider population. In addition, this study is limited by evaluation of these samples over only 1 year; yet, the impact of extended storage of samples to over 1 year needs to be studied. Finally, as different substances (for example bilirubin) can significantly affect measurements performed by the Jaffe method, evaluation of how these substances and protocols that try to minimize this assay interference 9, 10 are affected by long‐term storage needs to be incorporated in these type of studies.

CONCLUSION

UCr (but not SCr) measured by Jaffe are lower than samples measured by LC/MS. UCr measurements vary over time more when performed by LC/MS than Jaffe, mostly due to the sample measured after 1 year. Therefore, storage of urine for more than 90 days measured by LC/MS may provide altered results. Studies incorporating larger cohorts across a broader spectrum of CKD are needed to validate these results.