Clinical Biochemistry 43 (2010) 1158–1162 Contents lists available at ScienceDirect Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n b i o c h e m Development and validation of a rapid liquid chromatography isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for serum creatinine Pierre-Olivier Hétu ⁎, Marie-Ève Gingras, Bernard Vinet Department of Biochemistry, Hôpital Notre-Dame, Centre hospitalier de l'Université de Montréal, Montréal, Québec, Canada a r t i c l e i n f o Article history: Received 14 January 2010 Received in revised form 17 May 2010 Accepted 29 May 2010 Available online 8 June 2010 Keywords: Serum creatinine LC-MS/MS Isotope dilution Reference method a b s t r a c t Objectives: To develop an isotope dilution liquid chromatography tandem mass spectrometry (LC-IDMS/ MS) method for the standardization of serum creatinine. Design and methods: Supernatants obtained by protein precipitation were injected into a LC-MS/MS. Chromatography was performed on a cation exchanger pre-column in normal phase isocratic mode. Creatinine and creatinine-D3 were quantified using ion transitions of m/z 114→44 and 117→47, respectively. Results: The method was calibrated with the NIST Standard Reference Material 914a and was found to be exact in analyzing the certified reference material SRM 967 from NIST (97.1 ± 0.9% and 102.1 ± 0.9% of target value for levels 1 and 2, respectively) and by calculating recuperation of spiked creatinine in 10 different patient samples (103.6 ± 4.1%). Intra-assay imprecision was 0.9% at both 64.6 and 354 μmol/L creatinine, while inter-assay imprecisions were 1.9% and 1.8%. Absence of ion suppression was confirmed by spiking experiments. The method was shown to be free of carryover. A good correlation was obtained between the LCMS/MS method and a Jaffe method run on an automated analyzer (r = 0.999). Conclusions: We have developed a fast and simple method for the quantification of serum creatinine by isotope dilution tandem mass spectrometry (IDMS/MS) and we propose that this method can be used as a reference method by laboratories that wish to validate their serum creatinine automated assay. © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Introduction The prevalence of chronic kidney disease (CKD) is estimated at approximately 26 million people in the United States [1], and about 2.1 million in Canada [2]. Early identification and treatment of affected patients is critical because CKD is associated with increased morbidity and mortality, notably from cardiovascular complications [2–4]. The glomerular filtration rate (GFR) is the best marker of renal function. GFR cannot be measured directly, but it can be estimated by the clearance of inert exogenous compounds that are eliminated exclusively by filtration through the kidneys, such as inuline or 125I-iothalamate, or more easily by the clearance of endogenous compounds that are predominantly cleared by glomerular filtration, such as creatinine [5]. Creatinine is generated from creatine by normal muscular metabolism at a relatively constant rate, proportional to the muscle mass. Creatinine is mostly cleared from the blood by simple glomerular filtration but it is also Abbreviations: LC, Liquid chromatography; GC, Gas chromatography; MS, Mass spectrometry; MS/MS, Tandem mass spectrometry; IDMS, Isotope dilution mass spectrometry; HPLC-UV, High pressure liquid chromatography with ultraviolet absorbance detection; CKD, Chronic kidney disease; eGFR, Estimated glomerular filtration rate; SRM, Standard reference material. ⁎ Corresponding author. Biochimie, CHUM, Hôpital Notre-Dame, 1560 Sherbrooke Est, Montréal, Québec, Canada H2L 4M1. Fax: +1 514 412 7553. E-mail address: [email protected] (P.-O. Hétu). slightly secreted by the renal tubules, and this secretion can increase in compensation of reduced GFR [5]. Thus creatinine is not a perfect marker, but its clearance is nevertheless routinely used to evaluate GFR. However, measurement of the rate of creatinine clearance is laborious and error prone, mostly because of the necessity to properly collect urine during a 24 h period. In addition, creatinine clearance is usually only prescribed when the physician suspects a decreased kidney function. In an effort to better identify patients with CKD in early stages, it is now recommended to calculate and report an estimated GFR (eGFR) with every serum creatinine test result generated in the laboratory [6]. An eGFR reported for every patient, irrespectively of clinical signs or prior suspicion of decreased kidney function, should allow an earlier referral of CKD patients for proper follow up and potentially retard the progression of the disease. Multiple formulas exist for calculating eGFR from serum creatinine, including the Cockcroft–Gault [7], the MDRD (Modification of Diet in Renal Disease) [8,9], and the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) [10] formulas. However, the use of any of the formulas to estimate GFR requires exact and precise creatinine results. For example, a 7.6% under-estimation of the creatinine value will lead to an overestimation of eGFR of 9.6% when using the MDRD formula. It has thus been recommended that creatinine results used for calculating eGFR with the MDRD formula should be traceable to an isotope dilution mass spectrometric (IDMS) reference method, with desirable values for bias, imprecision, and total error of respectively 0009-9120/$ – see front matter © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2010.05.018 P.-O. Hétu et al. / Clinical Biochemistry 43 (2010) 1158–1162 3.4%, 2.2%, and 7.6% at approximately 88.4 μmol/L creatinine [6]. In line with this, most in-vitro diagnostic manufacturers have now calibrated their creatinine assays against an IDMS traceable method. Definitive methods for quantification of creatinine by gas chromatography coupled to isotope dilution mass spectrometry (GC-IDMS) have been described and they serve to assign creatinine values to the standard reference materials [11–14]. GC-IDMS methods have been certified by the Joint Committee for Traceability in Laboratory Medicine (JCTLM), an international committee in which participate the BIPM (Bureau International des Poids et Mesures), the IFCC (International Federation for Clinical Chemistry and Laboratory Medicine), and the ILAC (International Laboratory Accreditation Cooperation). However, the sample pretreatment prior to GC-IDMS analysis of creatinine is a very laborious multi-step procedure that includes precipitation of serum proteins, isolation of creatinine by liquid chromatography, evaporation of the creatinine fraction, and formation of creatinine derivatives. This complexity leads to higher costs per sample and to a lower throughput, which limits the applicability of definitive methods for large-scale validation of routine laboratory creatinine assays. Unfortunately, too few laboratories in the world offer the service of a definitive method for serum creatinine by GC-IDMS, which complicates the standardization of results. More recently, methods using liquid chromatography (LC-IDMS) have been developed [14–17], and such a method was used by the National Institute of Standards and Technology (NIST) to certify the creatinine concentrations in the two levels of their Standard Reference Material 967 [14]. Here we report the development and validation of a new simple liquid chromatography isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for creatinine quantification. The method is shown to be exact and precise, it is not affected by ion suppression effects, and it is free of sample carryover. In addition, the sample preparation is not complex and the method has a high sample throughput. These characteristics make the method a good candidate reference method that could be used to validate the IDMS traceability of routine clinical laboratory methods, and to reduce the differences that may subsist between different manufacturers' assays. Methods Materials Standard reference materials (SRM) of purified creatinine (SRM 914a) and creatinine in frozen human serum (SRM 967) were purchased from the NIST (National Institute of Standards and Technology, Gaithersburg, MD, USA). Deuterated creatinine (creatinine-D3, cat. #485446) and HPLC grade ammonium acetate, formic acid, and methanol were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). 1159 Sample processing Samples were prepared for analysis by transferring 15 μL of calibrator, control, or serum (using a 25 μL positive displacement pipette) to a 1.5 mL microcentrifuge tube containing 15 μL internal standard (10 μg/mL creatinine-D3 in water). Samples were then vortex mixed briefly and 1.2 mL of methanol was then added to precipitate the proteins. Samples were briefly vortex mixed, and after 10 min of mechanical stirring the samples were centrifuged at 11,000 × g for 5 min. Supernatants were transferred to vials and analyzed by LC-MS/MS. LC-MS/MS analysis Sample supernatants (5 μL) were injected on a 1200 series HPLC system coupled to a 6410 tandem mass spectrometer (Agilent Technologies). Normal phase chromatography of creatinine was performed on a strong cation exchanger SecurityGuard pre-column (SCX, 4 × 3 mm, 10 μm, cat. #AJ0-4308, Phenomenex, Torrance, CA, USA) at a temperature of 30 °C. Normal phase separation was achieved in isocratic mode at a flow rate of 0.6 mL/min using a mobile phase of 75% methanol and 25% of 10 mmol/L ammonium acetate containing 0.4% formic acid (v/v), in a total chromatographic run time of 3.0 min (with the first 0.3 min diverted directly to waste instead of the MS/MS detector). Detection of creatinine and creatinine-D3 was accomplished by tandem mass spectrometry in positive mode using multiple reaction monitoring with ion transitions of 114→44 and 117→47 m/z, respectively. Details of the mass spectrometry settings are listed in Table 1. The mass spectrometer was calibrated with 5 creatinine levels in duplicate, once at the beginning of the batch and once again at the end. The difference between duplicate curves was less than 1%, so the average of both curves was used for quantification of the controls and patient samples. Accuracy and precision Intra-assay precision was determined by quantifying creatinine in both levels of the standard reference material SRM 967 (5 times each), intercalated in a single series of 45 patient samples, while the values from 6 independent analytical series performed over 2 months were used to calculate the inter-assay precision. The accuracy was evaluated by comparing the average concentration of creatinine measured for both levels of SRM 967 with the NIST certified target values of 66.5 and 346.2 μmol/L, respectively [14]. In addition, the accuracy of the LC-IDMS/MS method was further validated by adding 10 μL of purified creatinine (8.95 mmol/L in 0.005 mol/L HCl) to 500 μL of different patient serums (n = 10, range of 31–338 μmol/L creatinine). The measured increase in creatinine concentration was compared to the expected increase of 175.5 μmol/L. Quantification of sample carryover Preparation of calibrators Creatinine standards were prepared gravimetrically. To prepare the 1 mg/mL primary standard, approximately 100 mg of SRM 914a was precisely weighed and transferred to a pre-weighed volumetric flask. The creatinine was then dissolved in 100 mL of HCl (0.005 mol/L), and the flask was weighed again. The concentration of creatinine in the primary standard was obtained by calculating the weight of creatinine using the certified purity of SRM 914a (99.8%) and the exact volume of water using a density of 0.997 g/mL (at 25 °C). The calculated concentration was converted to SI units (μg/mL× 8.84 = μmol/L). The highest calibrator for the calibration curve was then prepared gravimetrically by diluting the primary creatinine standard with water, and the other calibrators were obtained by serial dilutions with water using a recently calibrated positive displacement pipette. Sample carryover was evaluated according to the method of Broughton [18]. Briefly, two patient samples, one with high (H) and Table 1 Mass spectrometer parameters. Source parameters Gas temp: 350 °C Gas flow: 10 L/min Nebulizer: 50 psi Capillary voltage: 1200 V Acquisition parameters Fragments (m/z): Creatinine: 114→44 Creatinine-D3: 117→47 Fragmentor voltage: 100 V Dwell time: 50 ms Collision energy: 20 V 1160 P.-O. Hétu et al. / Clinical Biochemistry 43 (2010) 1158–1162 the other with low (L) creatinine concentration (837 μmol/L and 32 μmol/L, respectively), were sequentially analyzed in triplicate (H1, H2, H3 followed by L1, L2, L3). The percentage of sample carryover was calculated by the formula (L1 − L3) ÷ (H3 − L3) × 100, and the average of 3 such series was defined as the method's sample carryover. Evaluation of ion suppression The presence of ion suppression effects on the MS/MS response was evaluated by spiking 110.6 μmol/L creatinine in a pool of serum or in water (n = 5). The creatinine peak area corresponding to the added creatinine in the pool of serum was calculated by subtracting the endogenous creatinine from the total creatinine measured in the spiked serums. The MS/MS response, in absolute counts of peak area, calculated for the added creatinine in serum was then compared to that measured in water. Correlation with an automated method Results for patient samples analyzed by the LC-IDMS/MS method were compared to the results obtained from an IDMS traceable kinetic Jaffe colorimetric assay on an automated analyzer (Hitachi 917, Roche Diagnostics). All samples were analyzed in duplicate (n = 36) and the correlation between the methods was computed by Deming linear regression analysis using the CBstat version 5.1.1 software (K. Linnet, purchased from the AACC online store, cat. #2475, www.aacc.org). Table 2 Method accuracy (mean ± SD). Sample Target value (μmol/L) Measured value (μmol/L) % Recovery (measured/target) SRM 967, level 1 (n = 5) SRM 967, level 2 (n = 5) Spiked samples (n = 10) 66.5 (62.7–70.3) 64.6 ± 0.6 97.1 ± 0.9 346.2 (331.6–360.8) 354.5 ± 3.2 102.1 ± 0.9 175.5 181.8 ± 7.2 103.6 ± 4.1 Results A cost-effective, rapid, and simple LC-IDMS/MS method for the quantification of serum creatinine was developed. Even if a cation exchange chromatography material was used, the principal mechanism of separation is believed to be normal phase partition because the retention time of creatinine increased with increasing proportions of organic modifier in the mobile phase (data not shown). As expected, the higher methanol content in the mobile phase also resulted in a higher sensitivity on the mass spectrometer. In the chosen conditions, both creatinine and the deuterated internal standard eluted at approximately 1.6 min (Fig. 1A) in a total chromatographic time of 3 min. An injection overlap was also programmed, resulting in a throughput of approximately 17 samples per hour. Calibration of each analytical run was done in duplicate, at the start and at the end of the batch, and no drift in MS/MS relative response of creatinine/creatinine-D3 was observed between the duplicates (Fig. 1B). The calibrators showed a linear response on the mass spectrometer (from 34.6 to 553.3 μmol/L creatinine), although the data were better fitted by a second order polynomial function, as seen regularly in LC-MS. Accuracy and precision The proposed method was shown to be accurate by analyzing the standard reference material from the NIST (SRM 967, levels 1 and 2) and by spiking known amounts of creatinine in 10 patient samples (Table 2). The concentrations of creatinine measured for both levels of SRM 967 were accurate (97.1–102.1% of target value), and the added creatinine was completely recovered in the spiked patient samples. A higher variability was observed for the spiking experiments, partially because only a small volume of creatinine was added to the patient samples (to lessen the impact on the sample matrix), and also because the calculated recovery includes the imprecision of the pre- and post-spiking creatinine concentrations. The method was also very precise, with intra-assay precision below 1% and inter-assay precision below 2% for both levels of the NIST reference material (Table 3). Since creatinine-free serum samples were not available, the analytical sensitivity was determined using a sample with approximately 65 μmol/L creatinine (SRM 967, level 1), which is at the low end of the practical analytical range (i.e., the low end of the reference interval). The calculated analytical sensitivity, defined as three times the standard deviation observed for the SRM 967 level 1, was 1.7 μmol/L. The creatinine concentration of the lowest standard on the calibration curve (34.6 μmol/L) was defined as the lower limit of quantification, in accordance with published recommendations for chromatographic methods [19]. Table 3 Method precision. Sample Fig. 1. Typical chromatograph and calibration curve. A) Chromatograph of the NIST SRM 967 (level 1). The MS/MS response for creatinine (solid line, shaded peak) is overlaid on the response of the creatinine-D3 internal standard (dotted line). B) The 5 point calibration curve fitted with a second order polynomial equation. Intra-assay precision (n = 5) SRM 967, level 1 SRM 967, level 2 Inter-assay precision (n = 6) SRM 967, level 1 SRM 967, level 2 Mean (μmol/L) CV 64.6 354.5 0.89% 0.89% 64.7 353.3 1.9% 1.8% P.-O. Hétu et al. / Clinical Biochemistry 43 (2010) 1158–1162 Table 4 Ion suppression (n = 5). Sample Serum Water Endogenous creatinine Total creatinine (after spiking) Added creatinine (spiked) Added creatinine (spiked) Peak area (counts) CV 3.49 × 105 1.09 × 106 7.40 × 105 7.29 × 105 0.93% 1.58% 2.76% 1.75% Creatinine (110.6 μmol/L) was spiked in a pool of serum or in water. The peak area corresponding to the added creatinine in serum was calculated by subtracting the endogenous creatinine from the total creatinine concentration after spiking. Sample carryover and matrix effects The method was shown to be free of significant sample carryover by multiple sequential analyses of three high concentration samples followed by three low concentration samples (0.14% carryover, n = 3). The absence of an ion suppression effect on the MS/MS response was confirmed by spiking creatinine in a pool of serum or in water (Table 4). There was no significant difference between the spiked creatinine peak areas in water or serum (serum/water= 101.6 ± 2.9%, p = n.s.). Correlation with an automated method Finally, a routine IDMS traceable kinetic Jaffe method on the Roche Modular analyzer was compared with the LC-IDMS/MS method by analyzing 36 patient samples in duplicate with both methods (range of samples from 26 to 575 μmol/L creatinine). Deming regression Fig. 2. Correlation between LC-IDMS/MS and an automated kinetic Jaffe assay. A) The creatinine concentration in patient samples (n = 36) was determined in duplicate with both methods, and the results obtained with the automated assay were compared to those obtained by LC-IDMS/MS. Deming regression analysis results are presented, with 95% confidence interval of the slope depicted as dotted lines. B) Bland–Altman plot showing the relative difference between both methods, with the mean relative bias and the corresponding 95% confidence interval represented by dotted lines. 1161 analysis showed that the creatinine results obtained by the colorimetric assay correlated very well with the LC-IDMS/MS method (Fig. 2A), although a small but statistically significant constant bias of approximately 3.4 μmol/L was observed (p b 0.002). The calculated mean relative bias was 4.3% (Fig. 2B), and all but two data points fell within the 95% confidence interval (−5.8 to 14.5%). Discussion We present here the development and validation of a candidate reference IDMS method for the quantification of creatinine. The method is highly sensitive, it is exact and precise, it is free of significant sample carryover and it is not affected by ion suppression effects. Together with the relatively high throughput, achieved by the simple sample preparation and the sole use of a short cartridge for chromatography, these characteristics make the method ideal for the validation of the IDMS traceability of routine assays. Other mass spectrometric methods using deuterated creatinine as internal standard (IDMS methods) have been published [11–17]. Although the certified definitive methods are GC-IDMS methods [11– 13], LC-IDMS methods were recently described and have been used for assigning certified values to reference materials [14]. The LC-IDMS methods have the advantage of being much less labor intensive than GC-IDMS methods because derivatization of creatinine is required prior to GC analysis, which is not the case for LC-IDMS. A preliminary separation of creatinine from creatine by liquid chromatography must also be performed before GC analysis, since these two compounds become undistinguishable once derivatized [14,20]. The development of liquid chromatography based reference IDMS methods should thus be encouraged, in part because of the higher complexity and the lower throughput of GC-IDMS definitive methods compared to LC-IDMS, but mostly because results from both methodologies have been shown to be comparable [14,15]. Different strategies for the chromatography of creatinine can be employed in LC methods. Most previously published methods have used reversed phase [14–16] or ion exchange [17,21,22] chromatography. For our method, we chose to use a small cation exchange precolumn, as described previously by Owen et al. [17]. However, the method of Owen et al. was not calibrated nor validated using certified materials, and the intra- and inter-assay imprecisions were higher than those observed with the present method. Also, instead of the ion exchange chromatography proposed by Owen et al., normal phase partition chromatography retention of creatinine on the SCX cartridge was favored in our method by using a high proportion of methanol in the mobile phase. This strategy resulted in good retention of creatinine on the cartridge and was associated with very good sensitivity because of the higher volatility of the mobile phase that yields better ionization efficiency on the mass spectrometer. Additionally, it allowed the presence of higher proportions of methanol in the injected sample without affecting the chromatographic performance. The simple protein precipitation used for the pretreatment step was sufficient for sample clean-up, as previously published [15]. Thus, the proposed LC-IDMS/MS method for serum creatinine quantification is simple, accurate, precise, and sensitive, with a high throughput and a relatively low cost. The described method is proposed as a candidate reference method, because of its low imprecision (b2%) and its accuracy in the quantification of the certified reference material SRM 967. Unfortunately, it was not possible to compare our method with a definitive method before writing this paper because of the limited worldwide availability of certified GC-IDMS methods. The laboratory plays a significant role in the proper reporting of eGFR by assuring the production of reliable serum creatinine results, and this is capital for the correct identification and staging of CKD patients. In addition, the comparability of creatinine test results produced by different laboratories within a defined geographical region is important for the long term follow up of CKD patients, who 1162 P.-O. Hétu et al. / Clinical Biochemistry 43 (2010) 1158–1162 are likely to request laboratory tests from different hospitals. Although most manufacturers, if not all, now offer IDMS traceable creatinine assays, differences can still subsist between assays. Different hospitals have different creatinine assays from different manufacturers, so the comparability of the results between laboratories must be assessed, and eventually corrected, by external quality control and educational programs. For example, the successful implementation of a provincial creatinine standardization program in British Columbia (Canada), before the IDMS standardization of assays by instrument manufacturers, has been described [23]. This program, directed by the Canadian External Quality Assessment Laboratory (CEQUAL), consisted in sending GC-IDMS certified serum aliquots to all participating laboratories and by attributing proper correction factors to normalize the results of creatinine assays. With this strategy, the average bias and the average total error for the measurement of creatinine were reduced from 16.5 to 2.7% and from 23.9 to 8.7%, respectively [23]. The harmonization of creatinine results within a defined region, for example a province or a state, should be encouraged and the tools to achieve this goal need to be made available. To this end, the method presented here was developed to allow laboratories in the province of Quebec (Canada) to validate the IDMS traceability of their routine method, on a voluntary basis. The relatively low cost of the proposed LC-IDMS/MS reference method and the higher throughput compared to the definitive GC-IDMS methods should encourage the participation of laboratories to the provincial program, leading to more standardized creatinine results between different hospitals, to better estimation of GFR, and to better patient care. Acknowledgments The authors would like to thank, the SQBC (Société québécoise de biologie clinique) for financial support. The authors are also grateful to Dr M. Bouthiller (Hôpital de Granby, QC, Canada) for quantifying creatinine in patient samples with the Hitachi 917 analyzer and for providing these samples to allow comparison with our LC-IDMS/MS method. References [1] Coresh J, Selvin E, Stevens LA, et al. Prevalence of chronic kidney disease in the United States. JAMA 2007;298:2038–47. [2] Levin A, Hemmelgarn B, Culleton B, et al. Guidelines for the management of chronic kidney disease. CMAJ 2008;179:1154–62. 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