A Practical Example of CLSI C60 in Action: Vitamins and

1

A Practical Example of CLSI C60 in Action: Vitamins and Hormones

Lorin Bachmann, Ph.D., DABCCAssistant Professor, Pathology

Virginia Commonwealth University

[email protected]

AACC Conference-Mass Spectrometry in the Clinical Lab:Best Practice and Current Applications

Chicago, IL9/6/12

2

Disclosures

Thermo Fisher Scientific, Consulting

3

CLSI C60 “In Action” Presentation Notes

• Data shown was obtained BEFORE CLSI C60 draft guidelines were developed

• The presented data does not reflect draft guidelines in all cases,but will be used to frame discussions of draft CLSI C60 recommendations

• Draft CLSI C60 experimental design strategies are still underdevelopment and may not represent the final guidelines

• CLSI C60 Hierarchical Best Practice Recommendations will behighlighted, if available

4

CLSI C60 “In Action”: Topics to be Covered

Stability

Blank Matrix, S/N, LLoQ

Precision, Accuracy

Linearity, Dilution

Carryover

Matrix Effects

Interferences

Quality Assurance

5

CLSI C60 “In Action”: References

• Current Versions of Clinical and Laboratory Standards Institute(CLSI) Guidelines

• FDA Guidance for Industry: Bioanalytical Method Validation, May 2001

• European Union (EU) Directive 2002/647/EC: Analytical Methods and Interpretation of Results

• European Medicines Agency (EMA): Guideline on Bioanalytical Method Validation, Feb 2012

• Current CLIA Guidelines and CAP Checklist Items

• CLSI C60 Document Development Committee Consensus

6

C60: Assess analyte stability in native matrix under appropriate storage conditions

Short-term stability at RT (Bench top)Long-term stability at 2-8˚C, -20˚C or -70˚CMax # of Freeze/Thaw cycles, if applicable

CLSI C60 Recommendations: Stability Assessments

FDA Experimental Design:

• General: Assess bias, imprecision - stored vs. fresh samples3 aliq of 2 conc (Max 3x LLoQ and “near” ULoQ (EMA)),

• Short-term: RT for 4-24hr • Long-term: 3 storage durations• Freeze/thaw: Thaw unassisted, assess at least 3 F/T cycles

**CLSI Guideline Under Dev: C55, Protocols for Establishment of Sample Stability in Clinical Chemistry and Toxicology; Est release

June 2013

7

C60: Assess stability of reagents (extraction reagents, mobile phases, IS, stock solutions)

C60: Assess analyte stability during all phases of the analytical measurement process

• Monitor bias and imprecision of QC or other matrix approptest materials over duration of reagent storage

• Monitor for extraction efficiency changes over time• Monitor for degradation of IS signal, hydrogen-deuterium exchange

• Determine max bench-top processing time (ex: extracts at RT)• Determine max storage duration of extracts/preparations in the autosampler (evaporation effects, analyte degradation)

CLSI C60 Recommendations: Stability Assessments

**CLSI EP25 – Evaluation of Stability of In Vitro Diagnostic Reagents

8

Stability of Extracts Stored in Autosampler

25-OH D3Injected extracted cal, QC and native patient samples at T = 0Extracts re-injected after 17hr and 24hr storage in autosampler (2-8˚C)

Acceptability Criteria: ± 10% or ≤ 2 ng/mLSubset of N=36 patient samples shown

9

CLSI C60 Recommendation: Validation of a Blank Matrix

C60: Validate a blank matrix for use in subseq validation procedures (Cal prep, LLoQ, AMR, recovery…ect)

• Ensure low bkg noise in solvent/extraction reagent• Meas peak area of a double blank matrix (no analyte/no IS)

- Can use BSA or stripped serum if no analyte-free native matrix

No peak OR Blank resp < 20% peak area of LLoQ resp and <5% of IS resp

in 5-6 lots of blank material

C60 Best Practice Acceptability Criteria:

Testo; MeOH Testo; Stripped Serum Testo; 3 ng/dL289/109 289/109

10

CLSI C60 Recommendations: Signal-to-Noise (S/N)

CLSI EP17 LoD Limitations:

• LoD design is resource intense (40-60 reps/sample, 3-5 samples, 5 runs)

• LoD is not generally relevant for LC-MS/MS

C60: Calc S/N using blank matrix vs. matrix at LLoQ

S/N at LLoQ of 20:1 to ensure ruggedness


C60 Min Criteria

S/N at LLoQ of 10:1

11

S/N Verification at the LLoQ

Testosterone spiked into 2 lots of stripped serum N=8 independent extracts, analyzed over 2 runs

1.0 ng/dL 2.5 ng/dL 5.0 ng/dL

12

CLSI C60 Recommendations: Assessment of LLoQ & Precision

CV < 20% at LLoQ, < 15% bias if using a CRM

C60: Use CLSI EP17 to validate LLoQ


• 40 reps/sample, 3-5 samples/run over 5 runs

C60: Use CLSI EP5 to validate Precision

• 2 conc, 2 runs/d, in duplic for 20d• Calc Within-run (“Repeatability”) and Between-run (“Within-Laboratory”) CVs using ANOVA

C60 Best Practice Acceptability Criteria: CV ≤ 15% except at LLoQ where < 20% is acceptable

13

Precision Assessment (CLSI EP5-A2)

25-OH D3 Acceptability Criteria:CV < 10% or < 20% at LLoQ

25-OH D3(ng/mL) % CVwr % CVbr

1.0 7.1 15.818.8 3.2 5.338.9 2.7 3.9

[8% BSA][PP][PP]

14

Imprecision Profile

Imprecision profile generated to determine %CV across AMR25-OHD3 spiked into various matriciesAnalyzed in quadruplicate/run for 2 runs

Stripped Serum spiked with D3Native Patient Serum with endogenous D3

8% BSA spiked with D3

15

CLSI C60 Recommendations: Accuracy

C60: Assess accuracy using multiple approaches

C60 Best Practice (Exper described in CLSI EP15, EP9):Validate “trueness” using a “reference of higher order” (CLSI X5, ISO 17511) listed by JCTLM (approved RMPs, ref labs and RMs)

C60 Alternative Approaches:

• Method Comp vs. a JCTLM-approved RMP• Matrix-approp CRMs (pref commutable)

• Spike and Recovery – if RMP or RMs are unavail

• Accuracy-Based PT Materials (limited # of samples) • Method Comp vs. Previous Method (Bias vs. prev methodonly, not trueness)

Use native patient samples whenever possible Analyze a min of 40 patient samples in

duplicate over 5d (CLSI EP9-A2)

(limited # of samples, RMs not necessarily commutable)Described inCLSI EP15

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Accuracy: Method Comp with RMP - VDSP

TElimits

• ODS/CDC VDSP(www.ods.od.nih.gov/Research/VitaminD.aspx#vdsp)

• CDC HoST(www.cdc.gov/labstandards/hs.html)

**Suggested TEa based on published biological variation data

17

Accuracy: Certified Reference Material (CRM)

Single Run Analyzed9 replicates of NIST SRMs

Acceptability Criteria:±10% or 2 ng/mLbias vs. NIST

CLSI EP15-A2: 2 reps/run, 3-5 runs

Can use power calc to det# of reps required

18

Accuracy: Spike and Recovery

25-OH D3 Spiked into 8% BSA (Low Conc)25-OH D3 Spiked into Stripped Serum with back-calculationN = 10 reps/sample over 2 runs

8% BSA

C60 Best Practice:Spike into native matrix, 3 conc (L,M,H)/5 reps (FDA)

Stripped Serum

Acceptability Criteria:±10% or 2 ng/mL

19

Accuracy: Accuracy-Based PT Programs

Total 25-OHD, D2, D3 and D3-epi ref values providedNote that 3-epi D3 is NOT included in the Target Value

• CAP Accuracy Based Vitamin D Survey• CAP Testosterone and Estradiol Accuracy Survey

Value-assigned usingCDC RMPs

20

Bias vs. Previous Method (IA)

N = 167

Immunoassay (ng/mL)

LC-M

S/M

S (n

g/m

L)

Immunoassay (ng/mL)

% D

iff L

C-M

S/M

S v

s. IA

Total 25-OH D: LC-MS/MS vs. IA

21

25-OH D3: LC-MS/MS vs. 2 independent LC-MS/MS Methods

Bias vs. LC-MS/MS methodLC

-MS

/MS

(ng/

mL)

LC-M

S/M

S (n

g/m

L)

N = 42 N = 29

LC-MS/MS Method A (ng/mL) LC-MS/MS Method B (ng/mL)

- Compare to a RMP, if possible

22

CLSI C60 Recommendations: Linearity

C60 Linearity: Assess Linearity Using CLSI EP6

C60 Best Practice:

Use matrix-appropriate materialSerial dilutions should be avoided (prop of pipetting errors)

• 9-11 (min 5) points with 2-4 reps each • Evaluate linearity using the Polynomial Regression Method(EP6-A)

23

Linearity (CLSI EP6-A) – Polynomial Regression ApproachO

bser

ved

Con

c (n

g/m

L)

Nonlinearity should be assessed for clinical significance

25-OH D3 analyzed used matrix-appropriate commercial linearity materials

Coded Conc (ng/mL)

24

High Testosterone sample diluted with stripped serum

Coded conc (ng/dL)

Obs

con

c (n

g/dL

)Linearity (CLSI EP6-A) – Polynomial Regression Approach

25

CLSI C60 Recommendations: Validation of Dilution Protocol

C60: Assess Dilutions Within and Outside of AMR

• Assess dilution effects using Recovery Experiments

CLSI EP15-A2: 2-3 reps/sample, over 3-5 runs

• Use analyte free matrix as the dilution buffer

• Sample extracts should be diluted to avoid ME whendiluting unextracted serum

26

Validation of Dilution Protocol

Conc tested w/in and outside of AMRUsed Recovery Experiment, 2 reps each over a single runAcceptability Criteria: Recovery ±10% or 2 ng/mL

According to SOP 25-OH D3 > ULoQ (150 ng/mL), extracts are diluted 1:2


Recovery ±115%, CV ≤ 15%

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• Inject one or more blank samples immediately after high sample to validate a known limit (ex: highest physiol conc)

• Inject a blank sample after increasingly high conc samples to determine the carryover limit

C60: Also assess carryover for a high conc sample followed by blank (FDA, EMA)

CLSI C60 Recommendations: Validation of Carryover

C60: Assess carryover using CLSI EP10

• 3 conc (L,M,H), 10 reps/run, 1 run/d for 5d (**or 20 d for manuf)

• Seq: Mid, High, Low, Mid, Mid, Low, Low, High, High, Mid

28

Carryover Assessment – CLSI EP1025

-OH

D3

(ng/

mL)

CLSI EP10 Protocol

Sequence Number

Low: 24.3 ng/mLMid: 65.3 ng/mLHigh: 154.3 ng/mL

C60: Best Practice Acceptability Criteria

Carryover data from EP10 should be eval in context of TEa

29

CLSI C60 Recommendations: Carryover (High Sample vs. Blank)

C60 Best Practice Acceptability Criteria: No peak in blank or < 20% of LLoQ

High D3 patient sample

Blank Sample(8% BSA)

30

C60 Recommendations: Evaluation of Matrix Effects

Common causes of Ion Suppression/Matrix Effects in LC-MS/MS:

• Salts, phospholipids, protein content• Effect of binding proteins, pH, or ionic strength on extr efficiency• Free fatty acids, metabolites, other organic molecules

- Matrix effects are highly method-dependent (esp depend on sample prep)

Testo 289/97Relative Intensity: 1.2e5 cps

Testo 289/97Relative Intensity: 1.6e5 cps

PPT LLE

31

1) Pre vs. Post Extraction Spike and Recovery (Matuszewski/CLSI-C50)

C60 Best Practice Hierarchical Experimental Design Strategies:

C60 Recommendations: Evaluation of Matrix Effects

2) Matrix Mixing (CLSI EP7-A2) - Admixtures of patient sampleto characterize ME

3) T – column infusion experiment

% Matrix Effect = (B/A)*100% Extraction Efficiency = (C/B)*100% Process Efficiency = (C/A)*100

A – Neat Standard in SolventB – Samples Spiked POST-ExtractionC – Samples Spiked PRE-Extraction

N=6 indep matriciesrecommended (EMA)

32

Pre vs. Post-Extraction Spike and Recovery (CLSI-C50)

Set C Set ASet B

% Matrix Effect (B/A*100) 85.7% Extr Effic (C/B*100) 76.4% Process Effic (C/A*100) 65.5

C60 Best Practice Acceptability Criteria:• Eval based on TEa requirements(CLSI EP7 – partition TE into bias, imprecision and interference components)

10 ng/mL D3 spiked into Low Patient Pool (~1 ng/mL)

% Matrix Bias -14.3% Matrix Bias Corr for IS -2.3

33

Eval of Matrix Effects: Matrix Mixing (CLSI EP7)Experiment for purpose of validating surrogate matriciesBut, this approach can also be used to validate ME in patient samples

Native Patient Serum mixed in proportion with Stripped Serum or 8% BSAN=4 reps over a single run

- contribution from endogenous D3 in stripped serum removed

Native Patient Serum + Stripped Serum:

Native Patient Serum + 8% BSA:

34

Muller et al. J Chromatogr. B 773: (2002) 47-52

Evaluation of Matrix Effects: T - Infusion

Codeine

Glafenine

Protein PrecipitationD3 in MeOH infused + injection of extracted Blk Serum

D3 IS

??D3 T1

D3 T2

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Exogenous and Other Endogenous Interferences

• Reagents and Disposables

• Collection Tube Additives

• Hemolysis, Lipemia, Icterus

• Drugs/Metabolites

• Physiological or Disease-Associated Interferences

• Isobaric – Interferences resulting in shared product ions w/test analytes

• Isotopomeric – Interferences that share a same precursor and product ion

• Adducts – chemical modifications resulting in a shared precursor andproduct ion

36

CLSI C60 Recommendations: Interference Testing

Bile Salts inPBC patient

Testo and IS in MeOH was extracted using sialized tubes

25-OH D3T1

C60: Check for interferences from reagents & disposables

C60: Check for potential endogenous interferences

Testo 289/97

Testo 289/109

Testo ISInterference obsin IS transtition

25-OH D3IST2/T1 = -38% vs. Cal Curve

37

Interference from Collection Tubes

Collected into a Red-Top Tube Collected into a SST

- Interfer is obs in 289/97 and 289/109- Interfer is method/sample prep dependent

Testo 289/97 Testo 289/97

38

Spike and Recovery for Hemolysis

Interference from Preanalytical Phase: CLSI EP7

Patient Admixtures for Lipid InterferenceTesting

Note: Intralipid may contaminate the source

Using hemoglobin-equivhemolysate

39

Dr Russell Grant and Brian RappoldLabCorp inc.

[Slides 40 & 41]

40[M+H]+ Product Ion Spectra, m/z 109/97 ratio

+MS2 (361.20): 10 MCA scans from Sample 1 (TuneSampleID) of MT20110119084354.wiff (Heated Nebulizer) Max. 1.6e6 cps.

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6343.1

361.3

315.3

299.2

325.0

96.9 255.5109.0279.2121.3 267.2239.1

251.0175.1 307.0285.1223.2183.3157.4133.3 209.3 235.1129.082.9 107.0 301.3 317.4189.2 295.298.2 137.1 273.1 320.4125.171.457.0


60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

7.0e6

7.5e67.7e6 121.1

363.4

309.1 327.4267.4

97.0

269.0147.2241.1135.0 296.9251.1 281.0173.1159.2109.0 187.0 225.2 239.2

345.3209.2149.3119.1 183.2263.3189.2 253.3213.1 315.382.9 155.0 285.093.2 231.299.1 137.3 177.3

110.957.3 68.9


60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.8e6

3.0e6

3.2e6

3.4e6

3.6e6

3.8e6

4.0e6

4.2e6

4.4e6

4.6e6

4.8e6

5.0e6

5.2e65.4e6 361.3163.1

121.1

135.0 299.1343.1267.1145.1 283.2241.4 325.3189.4 313.1139.2107.2 307.2255.081.2 96.9 171.3 225.2 279.5123.0 249.1199.3156.9 317.1303.369.4 231.0


60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

7.0e6

7.5e6

8.0e6

8.5e6

9.0e6

9.5e6

1.0e7

1.1e7

1.1e7

1.2e7

1.2e7

1.3e7

1.3e7

1.3e7 97.1

109.0

287.1

123.183.2 269.3173.2 211.3 229.0144.9 185.578.9 133.1 251.1159.269.0 121.2 199.3

Aldosterone (361.2) 1.0 Cortisol (363.2) 2.0 Cortisone (361.2) 0.5 Androstenedione (287.2) 0.7 +MS2 (289.20): 10 MCA scans from Sample 1 (TuneSampleID) of MT20110119085654.wiff (Heated Nebulizer) Max. 1.5e7 cps.

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

9.0e6

1.0e7

1.1e7

1.2e7

1.3e7

1.4e7

1.5e7 97.1

109.0

289.2

122.9253.283.1 271.3

121.2 175.295.1 147.3106.9 187.3161.2 213.2132.9 201.369.0


60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.00

5.00e5

1.00e6

1.50e6

2.00e6

2.50e6

3.00e6

3.50e6

4.00e6

4.50e6

5.00e6

5.50e6

6.00e6

6.50e6

7.00e6

7.50e6

8.00e6

8.50e6

9.00e6

9.50e6

1.00e7

1.05e7

1.09e7 97.1

109.1

331.3

313.2

253.3 295.1271.0163.1122.9

175.183.0 145.2 187.2171.1137.195.1 120.9 211.1 237.2 255.1217.4 277.3 289.2191.3151.268.9


60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e697.0

109.0

315.3

297.1123.2

83.3 279.3255.5163.1145.1 173.495.2 215.2132.9 239.5189.3107.0 227.368.9


60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.8e6

3.0e6

3.2e6

3.4e6

3.6e6

3.8e6

4.0e6 255.2

159.2

215.281.0

161.1 291.3145.1

173.4199.2

147.295.0107.0

132.9119.0

93.1 273.2108.9130.9 185.4

175.171.2

189.2 203.267.0 83.2 136.9 229.5151.057.1 125.0 217.4 235.2165.2 249.2179.4 276.4207.1

Testosterone (289.2) 0.8 17-OHProgesterone (331.2) 0.9 Progesterone (315.2) 0.8 Dihydrotesterone (291.2) 0.8 +MS2 (347.20): 10 MCA scans from Sample 1 (TuneSampleID) of MT20110119092802.wiff (Heated Nebulizer) Max. 1.1e7 cps.

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.00

5.00e5

1.00e6

1.50e6

2.00e6

2.50e6

3.00e6

3.50e6

4.00e6

4.50e6

5.00e6

5.50e6

6.00e6

6.50e6

7.00e6

7.50e6

8.00e6

8.50e6

9.00e6

9.50e6

1.00e7

1.05e7

1.10e71.14e7 311.1

121.0

347.4269.1

175.1

293.097.1

147.0 173.2251.1135.1109.2

163.2 287.1189.2137.0253.2185.3 267.2

149.1 241.1215.2169.3 211.1 329.3283.0119.1227.4 243.2157.2 197.1106.999.3

201.381.2 131.0 275.3143.287.1 203.3191.2 219.3 249.2181.169.2 233.1 296.2257.0125.461.1


60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

5.8e6 121.0

347.2329.4

97.1

311.1293.1135.1

171.2147.1109.2 175.1

163.3159.2 269.2107.0 253.1187.1 229.283.0101.1 211.4119.1131.0 265.1241.1149.4 169.2 197.2 271.2193.1 223.1283.1201.3 237.387.1 137.2 247.2155.3 299.1181.169.1 285.0260.255.1 71.1 125.1 314.3


60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.8e6

3.0e6

3.2e6

3.4e6

3.6e6

3.8e6

4.0e697.1

109.1

347.1

311.2123.0 329.483.2 269.3253.1173.2 293.1 299.3187.2159.2121.0 145.095.3 225.3211.480.9 229.1 281.1106.8 317.5264.9241.4181.469.1 191.1


60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

1.7e6

1.8e6

1.9e6

2.0e6

2.1e6

2.2e6

2.3e6

2.4e6

2.5e697.1 109.2

331.3

123.2

177.3313.3295.3

107.2 163.2135.081.1 253.3173.2149.3119.283.2 271.2137.1 189.2185.2 199.3 277.1211.3

237.2 255.1197.3143.0 283.2209.067.0 221.3101.3 153.4 265.287.0 261.171.355.3 303.2245.0 316.4125.3

21-Desoxycortisol (347.2) 0.8 Corticosterone (347.2) 0.4 11-Deoxycortisol (347.2) 0.8 Deoxycorticosterone (331.2) 1

Interference Assessment: Ion Ratios and Isobaric Compounds

41

ALDO

CPD ECPD F

CORT

11D

21D

ANDE2

E1DOC

T

17OHP

DHEA

17OPN DHT

PG

PN

Chromatographic Resolution is Required

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time, min0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

Inte

nsity

, cps

7

34

29

46

40

20

1495

2711

32

21

2

22

4

828

39

26

30

2517

19

37

4210

3

13

44

31

12

24

38

33

47

45

41

15

18

166

23

48

51

35

1

504943

36

See Abstract: Steroid Interference Determination Versus Clinically Relevant Steroids – ASMS 2010 MP13 311*

42

QA Monitoring – Run Acceptability and Sample AcceptabilitySlope & Intercept PASS Range

% Calibrator Recovery (ex: ±10%)

r2 or SE of Calibrator Curve

Run-to-run IS Peak Area Recovery Criteria

IS Recovery Criteria for each sample

RT, Peak Resolution

Presence of Interfering Peaks or Absent Peaks

Peak Shape/Peak Symmetry

T2/T1 Ratio Criteria (CLSI-C50)

43

Reagent Lot Change – Patient Comps and Linearity (CAP)

Plus chromatography acceptability criteriaPlus QC acceptability criteria (native pt based)

Coded Conc (ng/mL)

Obs

Con

c (n

g/m

L)

44

Periodic Accuracy Monitoring – Use of CRM

1) Create Patient Poolsnear NIST SRM values

2) Calc Method Bias vs.NIST SRM

3) Apply a Correction Factor to Patient Poolsto trace their valuesto NIST-assignedvalues

Using Patient Pools traceable to NIST SRM 972 (25-OHD)

45

Q&A!

Documents

A Practical Example of CLSI C60 in Action: Vitamins and