Validation of a Bioanalytical ICP-MS Method for Quantification of Potassium in Human Urine

Monica Whitmire, Jennifer Ammerman, Jeff Biss, Patricia de Lisio, Chaoyang Huang, Anastasia Osredkar, Zachery Smith
MPI Research, 54943 North Main Street Mattawan, Michigan 49071

ABSTRACT
Purpose.
To validate a bioanalytical inductively coupled plasma mass spectrometry (ICP-MS) method for quantification of potassium in human urine. This method was validated using typical bioanalytical validation parameters of sensitivity, selectivity, linearity, carryover, intra/inter-day accuracy, and precision using quality controls (QC), matrix effects, and ruggedness. Stability parameters were also evaluated: stock solution, freeze/thaw, bench top, reinjection, and long term.

MATERIALS AND METHODS

Calculations.
Net Normalized Intensities: Net normalized intensities were used to calculate a linear fit calibration curve and unknown concentrations from the following equation. Solution Type Internal Standard Calibration Stock Solution

RESULTS
Table 3 Summary of the Validation Stability Results for Potassium in Human Urine

Condition Ambient Ambient Ambient Ambient Frozen (~ -20C) Ambient Freeze/Thaw Cycles Ambient

Duration 99 days 90 days 91 days 5 days 243 days 25 hours 8 cycles 72 hours

Methods.
Reference standards were solid potassium chloride (QCs) and aqueous potassium (curve). Unfiltered human urine was purchased from BioReclamation. Linearity was assessed using eight calibration standards prepared in water at the beginning and end of each run. The range was equivalent to 100 to 2000 g potassium/mL of urine. Pooled QC samples were prepared by fortifying urine at 200, 400, 600 g/mL, and 4000 g/mL (assayed at a 1:5 dilution). The final concentration of potassium in QC samples was adjusted to account for endogenous matrix levels. Fresh non-matrix QC samples were prepared in water on the day of use at 200, 800, and 1200 g/mL. A scandium internal standard (IS) solution was utilized for all standard and QC preparations. A water blank containing IS and a water double blank were included in every run. Matrix effects and selectivity were evaluated using human urine matrices from six different lots fortified at QC Low level. An Elan 6000 ICP-MS and a Thermo-Finnigan Element 2 High Resolution ICP-MS were used to analyze the prepared samples. where: Inet = Net intensity, Iu = Intensity of unknown at analyte mass, Iuis = Intensity of unknown at internal standard mass, Ib = Intensity of blank at analyte mass, Ibis = Intensity of blank at internal standard mass, (Ib/Ibis)* = The average of the two normalized blank intensities in two sets of the calibration curves. Amount of Potassium in Human Urine:

Quality Control Stock Solution Calibration Standards Pre-processed Samples Post-processed Samples (Reinjection Reproducibility Duration)

Results.
Blank preparations showed no interference for potassium. System suitability, carryover and matrix effects all met the acceptance criteria. Linearity was consistently demonstrated, with an R-Squared of >0.998. Recoveries for calibration standards and QCs were within 10015% (10020% for the LLOQ) of the nominal concentrations for all assessments. Freeze/thaw was demonstrated for eight cycles. Ruggedness was demonstrated using a different analyst and different instrument approach. Stock solution (100 g/mL) stability was demonstrated following 90 days ambient storage. Analyte in matrix stability was demonstrated following 25 hours ambient and 243 days -20C storage. Reinjection reproducibility was demonstrated following 72 hours ambient storage.

Figure 1 Representative Calibration Curve

80.0

Normalized intensity (K/Sc)

where: DF = Dilution Factor of sample, i.e. (10 mL / 0.050 mL = 200), Final Solution Concentration (g/L) is calculated by Microsoft Excel using the raw data exported from the ICP/MS software. Percent Relative Error (%RE):

70.0 60.0 50.0 40.0 30.0 20.0 10.0

y = 0.0364x + 0.0231 R = 0.9999

Conclusion.
This bioanalytical ICP-MS method was successfully validated for the quantification of potassium in human urine.

OBJECTIVES
The primary objective of this study was to validate an analytical method for the quantitative determination of potassium in human urine using ICP-MS technology. This method was to be validated in accordance with the FDA Guidance for Industry for Bioanalytical Method Validation, 21 CFR 58 Good Laboratory Practice (GLP) regulations, and current MPI Research standard operating procedures (SOPs). The MPI Research Quality Assurance Unit was assigned to review all phases of the study including, but not limited to, analyst GLP-compliant bioanalytical validation and ICP-MS training, instrument validation and calibration, protocol and amendments, routine laboratory inspections, draft, and final reports. The method was to be validated over a range of 100 g/mL to 2,000 g/mL. The method was also to be evaluated for its capability to quantify potassium in human urine samples that contain potassium up to 4,000 g/mL, after dilution. Stability was to be evaluated following frozen storage for 1, 3, and 8 months. Analyte Species Matrix Type Matrix Anticoagulant Matrix Source Method Type Preparation Procedure Sample Volume Percent Carryover LLOQ (g/mL) Accuracy and Precision ULOQ (g/mL) Selectivity Capability of Dilution (concentration tested, dilution factor) Short Term Ambient Temperature Stability in Matrix Freeze/Thaw Stability in Matrix Thermo Element 2 High Resolution ICP-MS (High Resolution Mode) 60 seconds 40 seconds 20 seconds 10 seconds 3 1 3 Long Term Storage Stability in Matrix Perkin Elmer Elan 6000 ICP-MS 60 seconds, 48 rpm 40 seconds, 48 rpm 20 seconds, 24 rpm 10 seconds, 24 rpm 3 1 100 Reinjection Reproducibility Duration
LLOQ = Lower limit of quantitation ULOQ = Upper limit of quantitation RSD = Relative Standard Deviation % RE = Percent Relative Error

RESULTS
Table 2 Summary of the Validation Results for Potassium in Human Urine Potassium Human Urine None, not applicable BioReclamation, Inc. ICP-MS Dilution 50 L < 20% for analyte < 5% for internal standard 100 15.0% RE of their respective nominal 15.0% RSD 2,000 6 lots 4,000 g/mL, up to a 1:5 dilution 25 hours 8 cycles 200, 600 g/mL: 31 days at -20C 200, 600 g/mL: 98 days at -20C 200, 600 g/mL: 243 days at -20C 100 g/mL: 90 days at ambient temperature 100,000 g/mL: 91 days at ambient temperature 0.5 g/mL: 5 days 10 g/mL: 5 days 72 hours

0.0 0 500 1000 1500 2000 2500

Equivalent Potassium in Urine Concentration (mg/mL)

The acceptance criteria for calibration curve were a minimum of 75% of calibration standards within 15.0% RE of the nominal concentration (20.0% RE at the LLOQ level) and a coefficient of determination (R2) 0.995.

MATERIALS AND METHODS

Standard Information.
Solid potassium chloride reference standard (RS) was obtained from National Institute of Standards and Technology (NIST) and was utilized for the preparation of all QC samples. Aqueous potassium RS was obtained from Ultra Scientific and was used for preparation of all calibration standards. Aqueous scandium was obtained from Ultra Scientific and was used as the IS. Both aqueous standards were traceable to NIST. All standards were stored ambient. QC samples were prepared in human urine at 200, 400, and 600 g/mL. The results were adjusted for endogenous levels of potassium prior to use in calculations. These QCs were stored frozen. QC samples were also prepared in acidified water at 200, 800, and 1200 g/mL fresh on the day of use. Calibration standards were prepared in volumes of 50 mL in a solution of 1% nitric acid in water. The calibration standards were prepared in acidified water at levels equal to 100, 200, 500, 800, 1,000, 1,200, 1,500, and 2,000 g/mL potassium in human urine. The standards were stored in polypropylene tubes at ambient temperature. Working IS (10 g/mL) was prepared by pipetting 0.050 mL of a 10,000 g/mL IS primary standard and 0.500 mL of nitric acid and diluting to volume with reagent water in a 50-mL volumetric tube.

CONCLUSIONS
A bioanalytical method was successfully validated for the quantification of potassium in human urine using an ICP-MS technique. Matrix effects and instrument drift were minimized using a scandium internal standard (IS). The samples were diluted using acidified water (1% nitric acid). For the quantification of accuracy and precision, QC samples results were adjusted for endogenous potassium found in the control urine used for the QC preparation. The standard curves were linear (R2 0.995) over an equivalent range of 100 to 2000 g/mL potassium in urine. The QC LLOQ, Low, Mid, High and Dilution (100, 200, 400, 600, and 4000 g/mL respectively) samples all met the acceptance criteria for intra-day and inter-day statistics (15.0% RE of their respective nominal values [20.0% RE at LLOQ], 15.0% RSD [20.0% RSD at LLOQ]). Selectivity was successfully demonstrated using 6 lots of human urine. Carryover in the double blank samples was <20.0% of the lowest analyte calibration standard and <5.0% in the lowest IS calibration standard. Freeze/thaw stability was successfully demonstrated after 8 cycles of 20 C storage. Matrix effect was monitored using 6 lots of blank human urine and water fortified with potassium at 200 g/mL (QC Low). The RSD of the matrix factors for potassium (analyte) and scandium (IS) were 15.0%, which met the acceptance criterion. Ruggedness was performed using a different analyst on a different day on a different ICP-MS instrument and met the acceptance criteria. Short-term stability was acceptable following 25 hours of ambient temperature exposure. Long-term stability was acceptable after 31, 98, and 243 days of -20 C storage. Stability of stock standard solutions was successfully demonstrated after at least 90 days of ambient storage. Reinjection reproducibility was successfully demonstrated following 72 hours of storage.

Sample Information.
Unfiltered control human urine was purchased from BioReclamation, Hicksville, NY, and was stored frozen at -20C.

Instrumentation and Analysis.

Table 1 Bioanalytical ICP-MS Instrument Parameters Instrument Parameters Wash Time: Sample Rinse Time: Sample Uptake Time: Analysis Time: Passes/Replicates: Readings/Replicates: Sweeps (runs)/Reading:

REFERENCES
Food and Drug Administration. (1978) 21CFR58 - Good Laboratory Practice for Nonclinical Laboratory Studies. Rockville, Maryland: US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research. Food and Drug Administration. (2001) Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. MPI Research, SOP: V0002930, Validation of Bioanalytical Assays. MPI Research, SOP: V0003924, Quality Control Samples for Bioanalytical Studies.

Stability Standard Solutions