As a company-wide quality metrics, ISR pass/fail and observations are continuously documented and updated on a quarterly basis.
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1 Appliction Note #2009-LC/MS/MS-001 >>> Incurred Smple Renlysis Introduction The Incurred Smple Renlysis (ISR) progrm hs been implemented in most bionlyticl lbortories s n dditionl mesure to evlute the relibility of the dt nd ssy performnce. A filed ISR requires n investigtion to determine why the ssy is not performing t the sme level during smple nlysis s compred to the initil method vlidtion runs. On the other hnd, successful ISR ssessment helps to improve confidence in the relibility of dt. Although QPS hs been performing ISR on some smples since 2003, we implemented compny-wide SOP on ISR s n dditionl mesure for qulity on Jnury 21, This ppliction note documents some of the ISR findings for LC/MS/MS ssy over 14 month period. Tbles 1 nd 2 re the most current metrics. Tble 1 represents n overview of the percentge ISR pss rte by the # of smples nd the # of studies within the first 14 months when ISR ws implemented t QPS compny-wide. Tble 2 is more detiled metrics ech br represents one study, nd the percent devition from the originl dt for ech ISR smple is grouped together into three distinct bnds within ech study. Tble 2 gives us cler visul metric of how mny smples pss ISR nd how close the ISR dt is to the originl dt. Two cse studies re lso presented to discuss some of the more interesting ISR observtions. As compny-wide qulity metrics, ISR pss/fil nd observtions re continuously documented nd updted on qurterly bsis. Tble 1. LC/MS/MS ISR Pss Rte (1/21/2008 4/3/2009) 100 % of ISR Pssed Out of 4059 Smples 92.2 Out of 84 Studies 80 % By the # of Smples % By the # of Studies LC/MS/MS Studies Tble 2. Stck Plot of % Devition from Originl Vlue (1/21/2008 4/3/2009) 100% % Smples In Study 66.67% 75% 50% 25% % >50% % >20% <50% % <20% 0% Study #
2 Cse Study 1 This is 4-in-1, prent nd metbolites, dog plsm ssy developed to support niml sfety studies. A stble isotope lbeled (SIL) ws used s n internl stndrd (IS) for the prent compound, A, nd n nlogue ws used s IS for the three metbolites (B, C, nd D). The ccurcy nd precision for ll four nlytes during method vlidtion rnged from -11.8% to 13.4% nd 1.4% to 14.9%, respectively. ISR Summry Compound # of ISR Smples # of Smples Within +/- 20% % Pss A B C D Investigtion Following the filed ISR test for metbolites B nd C, the smple nlysis ws put on hold nd n investigtion ws initited. Upon exmining the dt nd the study records, the following observtions nd conclusion were noted: Observtions Inference 1 The ISR dt for the prent compound A nd the metbolite D were in excellent greement with the originl dt (100% pssed criteri). The reproducibility for the metbolites B nd C ws concern. These suggested tht there ws no experimentl error from the originl runs nd the ISR runs. 2 For both metbolites B nd C, the ssy performnce ws better for qulity control (QC) smples thn for the study smples. The in-study precision nd ccurcy for metbolite B rnges from -11.1% to 1.4% nd 3.2% to 5.8% nd for metbolite C rnges from -1.5% to 3.0% nd 1.8% to 5.9%. These suggested tht there my be mtrix effect difference between the blnk mtrix plsm used to prepre the stndrds nd the QCs, nd the study smples mtrix. Overll, it indicted tht the mtrix effect presented in the study smples hs negtive impct to the ssy vribility for metbolites B nd C, but not for prent A nd metbolite D. One possible rtionle to ccount for mtrix effect difference cn be ttributed to the suitbility of the IS. One obvious solution to minimize the mtrix difference would be to revlidte the ssy with the SIL-IS for ll four nlytes. However, considering the time required for synthesis, revlidtion, nd the concern on the study smple stbility, n lterntive pproch is to dilute out the mtrix effect with the blnk mtrix used to prepre stndrd nd QCs with sufficiently lrge dilution fctor (e.g., 10). Although this pproch my be pplicble to metbolite B, it will not be pplicble to the minor metbolite C, s most smples fter 10-fold dilution will result in below quntifible limit (BQL) of metbolite C. With the sponsor s pprovl, the originl dt generted for metbolite C will only be used for informtion only. In order to test the bove hypothesis, some study smples were pooled to generte 20 individul smples. These pooled smples were ssyed twice for metbolite B using 10-fold dilution. The result of the dilution experiments showed tht the ssy vrition cused by mtrix in the study smple ws effectively minimized (Tble 3).
3 Tble 3. Dilution Experiments Showing the Mtrix Effect ws Minimlized for Metbolite B Anlyte B Investigtion Results # of INV smples % Pss Smple ID 1st Anlysis Run ID 1st Anlysis Conc. (ng/ml) 2nd Anlysis Run ID 2nd Anlysis Conc. (ng/ml) % Difference Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple Pooled Smple % Difference = ((1 st Ressy 2 nd Ressy)/Averge of 1 st Ressy nd 2 nd Ressy) x 100 Subsequently, metbolite B ws ressyed from ll smples, except from the control groups, with 10-fold dilution. The relibility of the newly generted dt for metbolite B ws supported by successful ISR test (90.5% pss). Tble 4. ISR Summry for Metbolite B fter Investigtion Metbolite B ISR Results fter Investigtion # of ISR smples % Pss Smple ID Originl Anlysis Run ID Originl Conc. (ng/ml) Repet Run ID Repet Conc. (ng/ml) % Difference PWL3F25 C Dy 7 1h PWL3F25 C Dy 7 2h PWL3F25 C Dy 7 6h PWL3F25 C Dy 7 12h PWL3F27 C Dy 7 2h PWL3F27 C Dy 7 4h PWL3F27 C Dy 7 6h PWL3F27 C Dy 7 12h PWLF328 C Dy 1 6h PWL3F28 C Dy 7 2h PWL4F32 D Dy 1 1h PWL4F32 D Dy 1 2h PWL4F32 D Dy 1 4h PWL4F32 D Dy 1 6h PWL4F32 D Dy 1 12h PWL4F32 D Dy 1 24h PWL4F32 D Dy 7 0h PWL4F32 D Dy 7 1h PWL4F32 D Dy 7 2h PWL4F32 D Dy 7 4h PWL4F32 D Dy 7 6h PWL4F32 D Dy 7 12h PWL4F32 D Dy 7 24h PWL4M13 D Dy 1 6h PWL4M13 D Dy 7 0h PWL4M13 D Dy 7 1h PWL4M13 D Dy 7 2h PWL4M13 D Dy 7 24h PWL4M14 D Dy 1 2h PWL4M14 D Dy 1 4h PWL4M14 D Dy 1 6h PWL4M14 D Dy 1 24h PWL4M14 D Dy 7 0h PWL4M14 D Dy 7 12h PWL4M14 D Dy 7 24h PWL4M15 D Dy 7 0h PWL4M15 D Dy 7 1h PWL4M15 D Dy 7 2h PWL4M15 D Dy 7 4h PWL4M15 D Dy 7 6h PWL4M15 D Dy 7 12h PWL4M15 D Dy 7 24h % Difference = ((1 st Ressy Originl)/Averge of Ressy nd Originl) x 100
4 >>> Incurred Smple Renlysis Findings >>> Mtrix effect presented in the study smples ws the root cuse of the ssy vribility for the metbolite B nd C uncovered by ISR test. >>> Hving pproprite IS, such s SIL-IS, is importnt to minimize the potentil mtrix effect presented in the study smples, which my not be present during vlidtion. >>> If the sitution llows, dilution cn be n effective pproch to minimize the mtrix effect presented in the study smples. >>> ISR ssessment provides dditionl nd importnt dt for the evlution of ssy performnce nd dt relibility. Cse Study 2 This cse study illustrtes the importnce of exmining the ISR results even if it meets the cceptnce criteri to help identify possible systemic errors. This is discreet ssy using stble isotope lbeled internl stndrd (SIL-IS) developed to support clinicl studies. The ccurcy nd precision for ssy during method vlidtion is -8.5% to 7.2% nd 1.3% to 15.0%, respectively. ISR Summry The ISR dt (Tble 5) met the cceptnce criteri. However, upon further review, it ws noted tht most of the dt (13 out of 14 smples) tht filed to meet the +/-20% criteri were from Run 1. Furthermore, 9 other smples in Run 1 were beyond +/-15%. In contrst, the dt from Runs 3 nd 11 mtched well with the corresponding dt from the ISR run (Run 12). These observtions brought the integrity of ll Run 1 dt into question.
5 >>> Incurred Smple Renlysis Tble 5. Compound X Incurred Smple Reproducibility Results 101,G2,D13,0h ,G2,D14,0.5h ,G2,D14,1.5h ,G2,D14,2h ,G2,D14,3h ,G2,D14,4h ,G2,D14,6h ,G2,D14,8h ,G2,D26,0h ,G2,D27,0h ,G2,D28,0h ,G2,D28,0.5h ,G2,D28,1h ,G2,D28,1.5h ,G2,D28,2h ,G2,D28,3h ,G2,D28,4h ,G2,D28,6h ,G2,D14,0.5h ,G2,D14,1h ,G2,D14,1.5h ,G2,D14,2h ,G2,D14,3h ,G2,D14,4h ,G2,D14,6h ,G2,D14,8h ,G2,D27,0h ,G2,D28,0h ,G2,D28,0.5h ,G2,D28,1h ,G2,D28,1.5h ,G2,D28,2h ,G2,D28,3h ,G2,D28,4h ,G2,D14,0.5h ,G2,D14,1h ,G2,D14,1.5h ,G2,D14,2h ,G2,D14,3h ,G2,D14,4h ,G2,D14,6h ,G2,D14,8h ,G2,D28,0h ,G2,D28,0.5h ,G2,D28,1h ,G2,D28,1.5h ,G2,D28,2h ,G2,D28,3h ,G2,D28,4h ,G2,D28,6h ,G2,D14,0.5h ,G2,D14,1h ,G2,D14,1.5h ,G2,D14,2h ,G2,D14,3h ,G2,D14,4h ,G2,D14,6h ,G2,D14,8h ,G2,D28,3h ,G2,D28,4h ,G2,D28,6h ,G2,D28,8h ,G2,D28,10h ,G2,D28,12h ,G2,D28,16h ,G2,29,24h ,G2,D14,0.5h ,G2,D14,1h ,G2,D14,1.5h ,G2,D14,2h ,G2,D14,3h ,G2,D14,4h ,G2,D14,6h ,G2,D14,8h ,G2,D28,0h ,G2,D28,0.5h ,G2,D28,1h ,G2,D28,1.5h ,G2,D28,2h ,G2,D28,3h ,G2,D28,4h ,G2,D28,6h % Difference = ((Ressy Originl)/Averge of Ressy nd Originl) x 100 # of ISR smples % Pss Smple ID Originl Anlysis Run ID Originl Conc. (ng/ml) Repet Run ID Repet Conc. (ng/ml) % Difference
6 >>> Incurred Smple Renlysis Investigtion To ddress the bove concern, n investigtion ws initited with the following points in mind: >>> All Run 1 dt were rejected. >>> All smples from Run 1 were ressyed in singlet. >>> The concentrtion dt from the ISR run (Run 12) were compred to the ressy run (Run 13) for ll 34 smples. >>> The pre-defined criteri re tht the ressy run will be considered vlid if more thn 2/3 of these 34 smples re within +/-20%. Tble 6 is the comprison of the ressyed dt nd the ISR dt, nd it clerly indicted tht the ressy run dt support the originl ISR dt. This lso indicted tht there ws n experimentl error from the originl Run (1). Tble 6. Compound X Incurred Smple Reproducibility Results Smple ID ISR Anlysis Run ID Originl Conc. (ng/ml) Repet Run ID Repet Conc. (ng/ml) % Difference 101,G2,D13,0h ,G2,D14,0.5h ,G2,D14,1.5h ,G2,D14,2h ,G2,D14,3h ,G2,D14,4h ,G2,D14,6h ,G2,D14,8h ,G2,D26,0h ,G2,D27,0h ,G2,D28,0h ,G2,D28,0.5h ,G2,D28,1h ,G2,D28,1.5h ,G2,D28,2h ,G2,D28,3h ,G2,D28,4h ,G2,D28,6h ,G2,D14,0.5h ,G2,D14,1h ,G2,D14,1.5h ,G2,D14,2h ,G2,D14,3h ,G2,D14,4h ,G2,D14,6h ,G2,D14,8h ,G2,D27,0h ,G2,D28,0h ,G2,D28,0.5h ,G2,D28,1h ,G2,D28,1.5h ,G2,D28,2h ,G2,D28,3h ,G2,D28,4h % Difference = ((Ressy Originl)/Averge of Ressy nd Originl) x 100 % Pss >>> >>> >>> Findings A successful ISR ssessment helps improve confidence on the performnce of the ssy nd relibility of dt, but it does not men tht the work required to ensure dt qulity is complete. Crefully exmining the ISR results, even if it meets the cceptnce criteri, my help identify possible systemic errors. It is importnt to look beyond the cceptnce criteri from n ISR to further improve the confidence of the dt. Delwre Technology Prk 3 Innovtion Wy, Suite 240 Newrk, DE web TEL (302) Fx (302)
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