CCQM-K12: Key Comparison on the Determination of Creatinine in Human Serum. Final Report March 2003

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1 CCQM-K12: Key Comparison on the Determination of Creatinine in Human Serum Final Report March 2003 Michael Welch, Curtis Phinney, Reenie Parris, and Willie May Analytical Chemistry Division Chemical Science and Technology Laboratory National Institute of Standards and Technology Gaithersburg, MD USA Gwi Suk Heo Korea Research Institute of Standards and Science Daejeon, South Korea Andre Henrion Physikalisch-Technische Bundesanstalt Braunschweig, Germany Gavin O Conner Laboratory of the Government Chemist Teddington, UK Heinz Schimmel Institute for Reference Materials and Measurements Geel, Belgium INTRODUCTION The accuracy and traceability for clinical laboratory measurements is becoming increasingly important. New regulations such as the EU IVD Directive are requiring that in vitro diagnostic products be traceable to measurement standards of a higher metrological order. To begin to address the need for higher order standards for clinical laboratory measurements, the Consultative Committee on the Amount of Substance (CCQM) is conducting a series of exercises to document the capabilities of NMIs in this area. Because there are hundreds of health status markers being measured in clinical laboratory settings, it is not possible to perform comparison exercises for all of them. To better focus the exercises, the Organic Working Group of CCQM decided to perform studies for three important health status markers that are representative of the measurement challenges associated with the quantitative determination of well-defined organic substances in blood. In , a Key Comparison (CCQM-K6) for the determination of cholesterol in serum was completed and the report is now available on the BIPM website 1. Cholesterol is present at relatively high concentrations in serum, is of very low polarity, and is predominantly esterified with fatty acids in blood. In 2001, two more Key Comparisons were conducted, one for serum glucose 2 (CCQM-K11) and one for serum CCQM-K12 Page 1 of 15

2 creatinine. Glucose is also present at relatively high concentrations in serum. It is highly water soluble and is partially associated with serum proteins. In contrast, creatinine, a small product of protein metabolism, is present at much lower concentrations. In addition, creatine, the openringed analog is also present. Without proper handling, creatine and creatinine can interconvert, leading to biased results. Because the array of challenges in accurately measuring these three analytes in human serum covers a wide range of challenges associated with measuring other organic molecules with molecular weights less than 500 daltons, a laboratory that can demonstrate competence for all three of these measurands has a basis for claiming competence for measurements of other organic compounds in the same range of concentrations and molecular weights in human serum. Creatinine is measured to determine renal function. It is formed by the spontaneous, nonenzymatic cyclization of creatine, a key component involved in muscle contractions. A rise in creatinine levels in blood may signify that the kidneys are not functioning as they should. Traditionally, creatinine was measured using a method that involved the Jaffe reaction, which is the reaction of creatinine with picrate ion to form a red color. The reagent cross-reacts with a number of substances, so various approaches are used to eliminate or correct for known interferences. In recent years, enzymatic assays, with or without using the Jaffe reaction, have become the methods of choice for routine measurements of creatinine in serum. As with glucose, different methods, different reagents, etc, may lead to significantly different results. Creatine, the open-ringed precursor of creatinine, may also react with reagents in some methods, causing a bias. Therefore, reference methods and materials are needed to maintain adequate accuracy in routine measurements for creatinine in blood. This report describes the 2001 Key Comparison, CCQM-K12, on measurement of serum creatinine. This project was approved by the CCQM after completion of a successful pilot study in 2000 (Table 1). The materials used for this study were from the same two lots of frozen serum materials used for the IMEP-17 interlaboratory comparison. The materials had been prepared under the direction of A. Uldall (DEKS, DK). Participants in this CCQM study received samples directly from the Institute for Reference Materials and Measurements (IRMM). The coordinating laboratory for this study, NIST, sent instructions and forms to the participants. The NMIs that agreed to participate in K12 were: Institute for Reference Materials and Measurements (IRMM) [EU] Korea Research Institute of Standards and Science (KRISS) [S. Korea] National Institute of Standards and Technology (NIST) [USA] Coordinating Laboratory Laboratory of the Government Chemist (LGC) [UK] NMi Van Swinden Laboratorium (NMi) [The Netherlands] Physikalisch-Technische Bundesanstalt (PTB) [Germany] Results were received from IRMM, LGC, NIST, PTB and KRISS. All of the participants used isotope dilution in their methods. Three of the participants (IRMM, NIST, and PTB) used methods based upon gas chromatography/mass spectrometry (GC/MS), while LGC and KRISS CCQM-K12 Page 2 of 15

3 used a method based upon liquid chromatography/mass spectrometry (LC/MS). Both approaches were approved for use in calculating the Key Comparison Reference Value based upon results from the pilot study. SUMMARY OF PILOT STUDY A pilot study (CCQM-P9) for the determination of serum creatinine was organized by NIST in Samples from two lyophilized human serum pools were sent to five participating institutions. These pools were unknown to the participants, except for the coordinating laboratory, and were the two levels of NIST SRM 909b Human Serum. Creatinine had previously been certified in these materials using the published isotope dilution/gas chromatography /mass spectrometry (ID/GC/MS) definitive method for serum creatinine 3. Each participant also received a sample of SRM 914a, Creatinine, to use for calibration. The participants were free to use whatever methods they chose. Results were received from all five participants, four of whom used ID/GC/MS-based methods, while the other (LGC) use an ID/LC/MS-based method. The results are shown in Table 1. The scatter in the data from the participants was significantly lower than what was observed for the pilot study for cholesterol in serum (CCQM-P7). Furthermore, there was excellent agreement between the mean values and the certified values for the two levels of SRM 909b. All of the participants results overlap with the certified ranges for this SRM. While some of the participants have provided results that suggest a systematic bias, the size of the bias is quite small. The results also indicated that it is possible to obtain good results for serum creatinine using LC/MS as well as using GC/MS. Based upon the excellent agreement of this study, the CCQM decided to proceed with a Key Comparison. PROTOCOL FOR KEY COMPARISON The CCQM-K12 study utilized two frozen serum materials provided as part of the IMEP-17 study. One of these materials had a creatinine level in the normal range for adults while the other had a level representative of an elevated concentration in adults. Both materials came as unknowns to all of the participants, including the coordinating laboratory, although an approximate target value was provided for each material. Three of the laboratories (NIST, IRMM, PTB) used ID/GC/MS-based methods. The three methods used very similar column chromatography procedures, involving cation exchange, to separate creatinine from creatine. Two of the laboratories (IRMM, PTB) converted creatinine to a trimethylsilyl derivative using the reagent MSTFA. NIST used 2,4-pentanedione and ethanol to derivatize creatinine 3. Two of the laboratories used LC/MS-based methods, which do not require derivatization of creatinine. The LC/MS-based method used by LGC did not require prior separation of creatine, as that separation occurs in the LC column. In contrast, KRISS elected to remove creatine by ion exchange chromatography prior to the LC/MS analysis. CCQM-K12 Page 3 of 15

4 The measurement equation used to calculate the creatinine mass fraction in mg/g of serum is dependent upon how the calibration is performed. If bracketing is used as in the published NIST method 3, the equation is as follows: C = [(I Sam - I Lo ) x (W Hi W Lo ) + W Lo ] M Lab (I Hi - I Lo ) x M Ser Where: C I Sam I Lo W Hi W Lo M Lab I Hi M Ser is the mass fraction of creatinine in the serum sample; is the unlabeled/labeled ion intensity ratio measured for serum sample; is the unlabeled/labeled ion intensity ratio for the lower ratio calibration standard; is the unlabeled/labeled mass ratio for the higher ratio calibration standard; is the unlabeled/labeled mass ratio for the lower ratio calibration standard; is the mass of the labeled creatinine added to the serum sample; is the unlabeled/labeled ion intensity ratio for the higher ratio calibration standard; is the mass of serum sample In addition to the ion intensity measurements, the other critical measurement is determining the mass of the reference compound used to prepare the calibration standards. This measurement requires careful weighing of a material that has a known purity and associated uncertainty. In contrast to the pilot study, no calibration material was supplied for the Key Comparison by the organizers. Thus, each participant was responsible for selecting a calibration material that had a stated purity and uncertainty, which were to be incorporated in calculations of concentration and uncertainty. Four of the laboratories (IRMM, KRISS, NIST, and PTB) used SRM 914a Creatinine from NIST with a certified purity of 99.7 mass % ± 0.3 mass %. LGC used an unspecified material with a certified purity of 99.8 mass % ± 0.2 mass %. RESULTS FROM KEY COMPARISON The results for the two materials are shown in Tables 2a and 2b. The mean values from the participants for Material I are in acceptable agreement (range 2.1%), while for Material II, the agreement is excellent (range < 1.0%). Key Comparison Reference Values (KCRV) and associated uncertainties: Based on guidelines established by the CCQM Organic Working Group, Key Comparison Reference Values are to be established based on results from study participants that had their method(s) validated through participation in the preceding Pilot Study. As IRMM did not participate in the relevant pilot, their results were not eligible to be included in the KCRV calculation. There were no outliers in the data submitted; consequently, all of the eligible data were used to calculate the KCRVs. For both Material I and Material II, it was recommended and accepted by the Organic Analytical Working Group that the KCRV be assigned as the mean ± U of the eligible results. That calculation yields a KCRV of ± µg/g for Material I, corresponding to a 95% confidence interval of to µg/g. The KCRV for Material II is ± µg/g, corresponding to a 95% confidence interval of µg/g to µg/g. It is likely that CCQM-K12 Page 4 of 15

5 systematic biases contribute significantly to the total biases of some of the participants results. Because all of the eligible results are included in calculating the KCRV, systematic biases in individual results would bias the KCRV. Therefore the KCRV may not be the best estimate of the true mass fraction of creatinine in the materials. However, even with results with systematic biases included in the calculations, the true mass fractions should fall within the 95% confidence intervals. The Tables of Equivalence, which enumerate the relationships among the results of the participants in this Key Comparison, are shown in Tables 3a and 3b. The graphs of equivalence are shown in Figures 1 and 2. Tables 4a-4e describe the uncertainty calculations as reported by each of the participants. Different components of uncertainty and different approaches to the calculation of standard uncertainty were used by the participants. Important factors that contributed significantly to the results from the GC/MS-based methods included the measurement precision, the purity of the reference compound used, equilibration between the labeled and unlabeled forms, and the degree to which creatine was not completely separated from creatinine prior to derivatization. One of the LC/MS-based methods (LGC) performed this separation on-line and no derivatization was performed so contributions from creatine were not an issue, but the other factors listed would be sources of uncertainty for this LC/MS-based method. The other LC/MS-based method performed the creatine separation prior to analysis, similar to the GC/MS methods. Unless there is some conversion of creatine to creatinine in the LC/MS analyses, the presence of creatine should not affect the results. DISCUSSION Creatinine is present in human blood at much lower levels than is cholesterol (5 12 mg/l vs mg/l) and is a small, polar molecule. Measurement using a GC/MS-based method requires a separation of creatinine from creatine, the open ringed analog of creatinine. Without this separation, any creatine present will be converted to the same derivative as creatinine. For GC/MS, derivatization is necessary to block the polar groups, which would otherwise prevent creatinine from passing through the GC column intact. LC/MS-based methods do not require derivatization, but the polarity of the molecule makes retention on most reverse phase columns difficult, thus making separation from impurities more of a challenge. Furthermore, the low molecular mass (113 daltons) makes it more prone to matrix interferences than one would find with larger molecules. In spite of these measurement challenges, the three laboratories that used GC/MS-based methods and the two laboratories that used LC/MS-based methods provided results that were in very good agreement for both materials, one of which was representative of a normal blood creatinine level, while the other had a level that would be indicative of an elevated creatinine concentration. CONCLUSIONS AND HOW FAR THE LIGHT SHINES? This Key Comparison study demonstrated that the participating NMIs could successfully measure serum creatinine at normal and elevated levels, using ID/MS-based methods, with CCQM-K12 Page 5 of 15

6 interlaboratory expanded uncertainties of less than 0.8%. These results combined with those of CCQM-K6 1, Determination of Cholesterol in Human Serum, and CCQM-K11 2, Determination of Glucose in Human Serum, were chosen to provide evidence for the capabilities of participating NMIs to measure a wide range of relatively small (non-protein) organic analytes in Human Serum. Cholesterol was chosen, and studied previously because it represents a lipophilic serum analyte. Glucose is highly water-soluble and also associates strongly with proteins. Creatinine is very polar, present at much lower levels than cholesterol and glucose, and its determination requires considerable care to assure separation from creatine, without interconversion between creatinine and creatine. Two NMIs, NIST and PTB, have participated in all three of these key comparisons with consistently good agreement with the KCRVs and the other participants. The results from this suite of key comparisons provide supporting evidence for measurement claims of these two NMIs related to a range of well-defined, small organic molecules in human serum. For other NMIs to make similar claims, they may wish to participate in bilateral studies or subsequent key comparisons to document their capabilities across this suite of compounds. REFERENCES: 1 CCQM-K6 Determination of cholesterol in serum. Report available at: 2 CCQM-K11 Determination of glucose in human serum. Report available at: 3 Welch, M.J., Cohen, A., Hertz, H.S., Ng, K.J., Schaffer, R., Van Der Lijn, P., White V, E., Determination of serum creatinine by isotope dilution mass spectrometry as a candidate definitive method, Anal. Chem., 58, (1986). CCQM-K12 Page 6 of 15

7 Table 1. Results of Pilot Study (CCQM-P3), Creatinine in Human Serum Units micrograms/gram Material A (SRM 909b Level I) Difference Lab Mean U from Cert (%) NIST PTB DGKC* KRISS LGC Overall Mea Std Dev CV(%) 1.25 U 0.10 Cert value / Diff (%) Units micrograms/gram Material B (SRM 909b Level II) Difference Lab Mean U from Cert (%) NIST PTB DGKC* KRISS LGC Overall Mea Std Dev CV(%) 0.80 U 0.50 Cert value / Diff (%) *Deutsche Gesellschaft für Klinische Chemie, Bonn, Germany CCQM-K12 Page 7 of 15

8 Table 2a. Results for CCQM-K12 Creatinine in Human Serum: Material I units: micrograms/gram Standard degrees of Participant Mean Uncertainty freedom k U IRMM KRISS LGC NIST PTB Mean excluding IRMM Range (%) 2.12 Std dev of mean Degrees of Freedom 3 k factor U U(rel) % KCRV µg/g ± µg/g Table 2b. Results for CCQM-K12 Creatinine in Human Serum: Material II units: micrograms/gram Standard degrees of Participant Mean Uncertainty freedom k U IRMM KRISS LGC NIST PTB Mean excluding IRMM Range (%) 0.97 Std dev of mean Degrees of Freedom 3 k factor U U(rel) % KCRV µg/g ± µg/g CCQM-K12 Page 8 of 15

9 Table 3a. Matrix of Equivalence Material I MEASURAND: mass fraction of creatinine in human serum Material I (physiological range) The key comparison reference value, x R, is calculated as the mean of the participant results, excluding IRMM: x R = µg/g. The expanded uncertainty, U R, of x R, at a 95 % level of confidence, is: U R = µg/g. The degree of equivalence of each laboratory with respect to the reference value is given by a pair of terms: D i = (x i - x R ) and U i, its expanded uncertainty corresponding to a 95% confidence interval, both expressed in mg/g. The degree of equivalence between two laboratories is given by a pair of terms: D ij = D i - D j = (x i - x R ) - (x j - x R ) = x i - x j and U ij, its expanded uncertainty corresponding to a 95% confidence interval, both expressed in mg/g. Lab j Lab i IRMM KRISS LGC NIST PTB D i U i µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g IRMM KRISS LGC NIST PTB Table 3b. Matrix of Equivalence Material II MEASURAND: mass fraction of creatinine in human serum Material II (elevated range) The key comparison reference value, x R, is calculated as the mean of the participant results, excluding IRMM: x R = µg/g. The expanded uncertainty, U R, of x R, at a 95 % level of confidence, is: U R = µg/g. The degree of equivalence of each laboratory with respect to the reference value is given by a pair of terms: D i = (x i - x R ) and U i, its expanded uncertainty corresponding to a 95% confidence interval, both expressed in mg/g. The degree of equivalence between two laboratories is given by a pair of terms: D ij = D i - D j = (x i - x R ) - (x j - x R ) = x i - x j and U ij, its expanded uncertainty corresponding to a 95% confidence interval, both expressed in mg/g. Lab j Lab i IRMM KRISS LGC NIST PTB D i U i µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g IRMM KRISS LGC NIST PTB CCQM-K12 Page 9 of 15

10 Table 4a. IRMM - Uncertainty Reporting Form Material 1 Contributions to Uncertainty Type Relative Standard Degrees of (describe) (A or B) Uncertainty (%) Freedom standard solution B 0.16 mass sample B 5.5 * 10-3 Response factor A results A limit of detection B 1.20 Combined Relative Standard Uncertainty (%) Calculated Degrees of Freedom Coverage Factor (k) Relative Expanded Uncertainty (%) Mean Result in µg/g Expanded Uncertainty (U) in µg/g Material 1I Contributions to Uncertainty Type Relative Standard Degrees of (describe) (A or B) Uncertainty (%) Freedom standard solution B 0.16 mass sample B 1.0 * 10-2 Response factor A results A limit of detection B 1.20 Combined Relative Standard Uncertainty (%) Calculated Degrees of Freedom Coverage Factor (k) Relative Expanded Uncertainty (%) Mean Result in mg/g Expanded Uncertainty (U) in mg/g CCQM-K12 Page 10 of 15

11 4b. KRISS Uncertainty Reporting Form Material I Serum 1 Parameter Value unc v ci [ci*u)^2 (ci*ui)^4/v Wsample (g) large included in within sample varaiation Wis-sol, sample(g) large Wis-sol, std (g) large included in between sample varaiation Ws-sol, std (g) large Cs-sol (ug/g) large a- preparation repeatability (%) included in between sample varaiation b-creatinine weight (%) not included in between sample variation c-purity(%) not included in between sample variation b+c Arsample,corr included in between sample varaiation Arstd,corr var. between sample(ave) E-07 Conc. in Sample(ug/g) *Combined uncertainty of the measurement result u c =[(σ L ) 2 + (c i u i ) 2 ] 1/2., ci and u i are the sensitivity coefficient and the standard uncertainty of C s-sol (absolute). * The effective degrees of freedom of the combined uncertainty u c is obtained from the Welch-Satterthwaite formula. Material II Serum 2 Parameter Value unc v ci [ci*u)^2 (ci*ui)^4/v Wsample (g) large included in within sample varaiation Wis-sol, sample(g) large Wis-sol, std (g) large included in between sample varaiation Ws-sol, std (g) large Cs-sol (ug/g) large a- preparation repeatability (%) included in between sample varaiation b-creatinine weight (%) not included in between sample variation c-purity(%) not included in between sample variation b+c Arsample,corr included in between sample varaiation Arstd,corr var. between sample(ave) E-06 Conc. in Sample(ug/g) *Combined uncertainty of the measurement result u c =[(σ L ) 2 + (c i u i ) 2 ] 1/2., ci and u i are the sensitivity coefficient and the standard uncertainty of C s-sol (absolute). CCQM-K12 Page 11 of 15

12 Table 4c. LGC - Uncertainty Reporting Form Material 1 Contributions to Uncertainty Type Relative Standard Degrees of (describe) (A or B) Uncertainty (%) Freedom Precision of method (p m ) A Balance linearity (m x ) B Large Balance linearity (m z ) B Large Balance linearity (m y ) B Large Balance linearity (m yc ) B Large Conc. of calib. solution corrected B 0.12 Large for purity of 99.8% ± 0.2 % (C calib ) Combined Relative Standard Uncertainty (%) 0.2 Calculated Degrees of Freedom Coverage Factor (k) 2 Relative Expanded Uncertainty (%) 0.4 Mean Result in µg/g Expanded Uncertainty (U) in µg/g Material II Contributions to Uncertainty Type Relative Standard Degrees of (describe) (A or B) Uncertainty (%) Freedom Precision of method (p m ) A Balance linearity (m x ) B Large Balance linearity (m z ) B Large Balance linearity (m y ) B Large Balance linearity (m yc ) B Large Conc. of calib. solution corrected B 0.12 Large for purity of 99.8% ± 0.2 % (C calib ) Combined Relative Standard Uncertainty (%) 0.17 Calculated Degrees of Freedom Coverage Factor (k) 2 Relative Expanded Uncertainty (%) 0.34 Mean Result in µg/g Expanded Uncertainty (U) in µg/g CCQM-K12 Page 12 of 15

13 Table 4d. NIST - Uncertainty Reporting Form Material I Relative Uncertainty (%) Uncertainty type d.f. Steps in Process A B Purity of reference standard X inf Equilibration X inf Separation of creatine X inf GC/MS measurements X combined rel std uncertainty (%) Calculated degrees of freedom 3.9 k-factor Relative expanded uncertainty (%) Mean value 8.28 microgram/g Abs. expanded uncertainty 0.10 microgram/g Material II Relative Uncertainty (%) Uncertainty type d.f. Steps in Process A B Purity of reference standard X inf Equilibration X inf Separation of creatine X inf GC/MS measurements X combined rel std uncertainty (%) Calculated degrees of freedom 3.9 k-factor Relative expanded uncertainty (%) Mean value microgram/g Abs. expanded uncertainty 0.23 microgram/g CCQM-K12 Page 13 of 15

14 Table 4e. PTB - Uncertainty Reporting Form Material I Contributions to Uncertainty Type Relative Standard Degrees of (describe) (A or B) Uncertainty (%) Freedom Measurement SD of mean 1 A Purity of neat ref matl 2 B 0.17 inf. Systematic error in balance calibration 2 B inf. Systematic error in measurement procedure 2 B inf. Combined Relative Standard Uncertainty (%) 0.35 Calculated Degrees of Freedom 1429 Coverage Factor (k) Relative Expanded Uncertainty (%) Mean Result in µg/g Expanded Uncertainty (U) in µg/g Material II Contributions to Uncertainty Type Relative Standard Degrees of (describe) (A or B) Uncertainty (%) Freedom Measurement SD of mean 3 A Purity of neat creatinine reference material 4 B 0.17 inf. Systematic error in balance calibration 4 B inf. Systematic error in measurement procedure 4 B inf. Combined Relative Standard Uncertainty (%) Calculated Degrees of Freedom 35 Coverage Factor (k) Relative Expanded Uncertainty (%) Mean Result in µg/g Expanded Uncertainty (U) in µg/g This includes all type A components caused by weighing, spiking, sample preparation, GC/MS- measurement and heterogeneity 2 Supposed to be rectangularly distributed 3 This includes all type A components caused by weighing, spiking, sample preparation, GC/MS- measurement and heterogeneity 4 Supposed to be rectangularly distributed CCQM-K12 Page 14 of 15

15 Figure 1. Graph of Equivalence for Material I CCQM-K12 Creatinine in Human Serum (material I) Degrees of equivalence [D i and expanded uncertainty (95% confidence interval) U i ] 0.4 [Di = (x i - x R )]/µg/g x R = µg/g U R = µg/g IRMM KRISS LGC NIST PTB Figure 2. Graph of Equivalence for Material II CCQM-K12 Creatinine in Human Serum (material II) Degrees of equivalence [D i and expanded uncertainty (95% confidence interval) U i ] [Di = (x i - x R )]/µg/g x R = µg/g U R = µg/g IRMM KRISS LGC NIST PTB CCQM-K12 Page 15 of 15