Development and Validation of a Methodology for Analyzing ...€¦ · The extraction of MX-01 from...
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Covance is the drug development business of Laboratory Corporation of America ® Holdings (LabCorp ® ). Content of this material was developed by scientists who at the time were affiliated with LabCorp Clinical Trials or Tandem Labs, now part of Covance. Development and Validation of a Methodology for Analyzing Concentrations of MX-01 in Human Dried Blood Spot (DBS) Samples Deupree SM 1 , Omofoye FA 1 , Nofsinger JB 1 , Allen MS 1 , Liao D 2 , Paton M 2 and Qian M 2 1 Tandem Labs, Laboratory Corporation of America ® Holdings 2 Takeda Pharmaceuticals International Company Example Chromatograms – Matrix Controls Example Chromatograms – LLOQ AND ULOQ Example Chromatograms – Selectivity Blanks and LLOQ Spikes (Representative Individual Matrix Lot) Method Summary Method Development Methodology An accurate and precise methodology was developed and validated for quantifying the small molecule MX-01 in human dried blood spot (DBS). Calibrators and QCs were prepared in human whole blood (K2EDTA, adjusted to 45% hematocrit) and spotted (20 uL) onto Whatman DMPK-B cards using a positive displacement pipette. A laboratory grade punch tool of sufficient diameter to excise the entire DBS spot was not readily located. Instead, the DBS spot was excised using a 7/16” punch gasket purchased from a retail vendor. Extraction was effected by addition of an aqueous MX-01-d10 internal standard followed by protein precipitation. Separation from matrix components was achieved using a Waters BEH C8 column (2.1 x 50 mm, 1.7 um) operated at 40°C and an isocratic elution profile with a mobile phase composed of 17/83 (20 mM ammonium formate + 0.1% formic acid)/acetonitrile flowing at 0.4 ml/min on an Acquity ® UPLC (Waters). Peaks eluted between 1.0 – 1.1 minutes with a total run time of 2 minutes. A triple quadrupole mass spectrometer (AB Sciex ® API 5000) was used for detection using electrospray ionization in positive ion mode with multiple reaction monitoring. Methodology continued Results Results Summary Conclusions Excision and extraction of the entire DBS spot was found to significantly improve assay accuracy and precision as well as the ability to accurately quantify MX-01 in DBS samples prepared with a wide range of blood hematocrit percentages. Extraction of the entire spot may prove to be important for some DBS assays, particularly if some level of blood cell binding occurs; a thorough investigation could yield other factors that improve method performance. TROUBLE-SHOOTING PRECISION AND REPEATABILITY The primary challenge encountered during method development was achieving acceptable precision and repeatability. Furthermore, accuracy at hematocrit extremes did not initially meet acceptance criteria. A number of experimental parameters were tested. ■ Matrix effects – The was tested in real time by injection of extracted blank matrix coupled with post-column infusion of analyte – no enhancement/ suppression noted. ■ Pipetting technique – Three spotting techniques were compared for intra-pipetting precision (air- displacement, positive pressure, robotic liquid handler) – no significant difference was noted. ■ Whole blood age – Response factor for QCs quantified using curves prepared in four lots of blood received periodically over the course of a month were compared – no significant difference was noted. ■ Extraction variables – Various organic solvents and extraction steps were varied – no significant improvement was noted. ■ Excision technique – Partial versus whole spot excision was compared – a significant improvement in accuracy and precision were noted. The one factor that made the greatest difference toward improving performance was excision and extraction of the entire DBS spot rather than a 10 mm diameter portion. SAMPLE PREPARATION AND EXTRACTION ■ Calibrators are prepared as DBS within 24 h of extraction (fresh) and are allowed to dry on the benchtop at room temperature for 3 – 24 h ■ Excise entire DBS spot using a 7/16” punch gasket and hammer or mallet ■ Add 0.300 mL internal standard working solution (5.00 ng/mL each of the isotopically-labeled internal standard MX-01-d10 in water) and mix vigorously 5 min ■ Sonicate for 15 min. ■ Add 1.5 mL methanol to elicit protein precipitation, shake 15 min at 1000 rpm, and centrifuge at 0-8 °C 3000 RCF for 10 min ■ Using a Hamilton Star robotic liquid handling system, transfer 400 uL of supernatent to fresh plate ■ Dry under nitrogen at 45 °C ■ Add 200 uL of 20 mM ammonium formate + 0.1% formic acid using robotic liquid handling system ■ Cap and vigorously mix for 5 min at 1000 rpm to reconstitute ■ Centrifuge at 3000 RCF for 5 min ■ Store supernatant at 0-8 °C until analysis DBS Cards Drying DBS Extraction Instrumental Conditions (UPLC) Example Chromatograms – Carryover Calibrator Performance Curve Performance Over Three Core Accuracy and Precision Validation Runs Results Calibrator Performance Intra-Assay Accuracy and Precision of Quality Control Samples Inter-Assay Accuracy and Precision of Quality Control Samples from ANOVA Instrumental Conditions (MS) The extraction of MX-01 from DBS was validated over the range of 1.00 to 1,000 ng/mL with a dilutional linearity limit of 40,000 ng/mL. Precision, accuracy, and selectivity met validation acceptance criteria. Storage stability was demonstrated for 201 d at room temperature (pending additional experiments), 6 days at -70°C, and 3 days at 45°C. Accurate quantitation was noted in both hemolyzed and hyperlipidemic samples. Furthermore, when the entire punch was excised and extracted during validation, MX-01 was accurately and precisely quantified in spots prepared with hematocrit levels between 20 and 55%.
Transcript of Development and Validation of a Methodology for Analyzing ...€¦ · The extraction of MX-01 from...
Covance is the drug development business of Laboratory Corporation of America® Holdings (LabCorp®). Content of this material was developed by scientists who at the time were affiliated with LabCorp Clinical Trials or Tandem Labs, now part of Covance.
Development and Validation of a Methodology for Analyzing Concentrations of MX-01 in Human Dried Blood Spot (DBS) SamplesDeupree SM1, Omofoye FA1, Nofsinger JB1, Allen MS1, Liao D2, Paton M2 and Qian M2
1Tandem Labs, Laboratory Corporation of America® Holdings2Takeda Pharmaceuticals International Company
Example Chromatograms – Matrix Controls
Example Chromatograms – LLOQ AND ULOQ
Example Chromatograms – Selectivity Blanks and LLOQ Spikes (Representative Individual Matrix Lot)
Method Summary Method Development
Methodology
An accurate and precise methodology was
developed and validated for quantifying the small
molecule MX-01 in human dried blood spot (DBS).
Calibrators and QCs were prepared in human whole
blood (K2EDTA, adjusted to 45% hematocrit) and
spotted (20 uL) onto Whatman DMPK-B cards using
a positive displacement pipette. A laboratory grade
punch tool of suffi cient diameter to excise the
entire DBS spot was not readily located. Instead,
the DBS spot was excised using a 7/16” punch
gasket purchased from a retail vendor. Extraction
was effected by addition of an aqueous MX-01-d10
internal standard followed by protein precipitation.
Separation from matrix components was achieved
using a Waters BEH C8 column (2.1 x 50 mm, 1.7 um)
operated at 40°C and an isocratic elution profi le
with a mobile phase composed of 17/83 (20 mM
ammonium formate + 0.1% formic acid)/acetonitrile
fl owing at 0.4 ml/min on an Acquity® UPLC (Waters).
Peaks eluted between 1.0 – 1.1 minutes with a total
run time of 2 minutes. A triple quadrupole mass
spectrometer (AB Sciex® API 5000) was used for
detection using electrospray ionization in positive
ion mode with multiple reaction monitoring.
Methodology continued Results
Results Summary
ConclusionsExcision and extraction of the entire DBS spot was found to signifi cantly improve assay accuracy and precision as well as the ability to accurately quantify MX-01 in DBS samples prepared with a wide range of blood hematocrit percentages. Extraction of the entire spot may prove to be important for some DBS assays, particularly if some level of blood cell binding occurs; a thorough investigation could yield other factors that improve method performance.
TROUBLE-SHOOTING PRECISION AND REPEATABILITY
The primary challenge encountered during method development was achieving acceptable precision and repeatability. Furthermore, accuracy at hematocrit extremes did not initially meet acceptance criteria. A number of experimental parameters were tested.
■ Matrix effects – The was tested in real time by injection of extracted blank matrix coupled with post-column infusion of analyte – no enhancement/suppression noted.
■ Pipetting technique – Three spotting techniques were compared for intra-pipetting precision (air-displacement, positive pressure, robotic liquid handler) – no signifi cant difference was noted.
■ Whole blood age – Response factor for QCs quantifi ed using curves prepared in four lots of blood received periodically over the course of a month were compared – no signifi cant difference was noted.
■ Extraction variables – Various organic solvents and extraction steps were varied – no signifi cant improvement was noted.
■ Excision technique – Partial versus whole spot excision was compared – a signifi cant improvement in accuracy and precision were noted.
The one factor that made the greatest difference toward improving performance was excision and extraction of the entire DBS spot rather than a 10 mm diameter portion.
SAMPLE PREPARATION AND EXTRACTION■ Calibrators are prepared as DBS
within 24 h of extraction (fresh) and are allowed to dry on the benchtop at room temperature for 3 – 24 h
■ Excise entire DBS spot using a 7/16” punch gasket and hammer or mallet
■ Add 0.300 mL internal standard working solution (5.00 ng/mL each of the isotopically-labeled internal standard MX-01-d10 in water) and mix vigorously 5 min
■ Sonicate for 15 min.■ Add 1.5 mL methanol to elicit protein
precipitation, shake 15 min at 1000 rpm, and centrifuge at 0-8 °C 3000 RCF for 10 min
■ Using a Hamilton Star robotic liquid handling system, transfer 400 uL of supernatent to fresh plate
■ Dry under nitrogen at 45 °C■ Add 200 uL of 20 mM ammonium
formate + 0.1% formic acid using robotic liquid handling system
■ Cap and vigorously mix for 5 min at 1000 rpm to reconstitute
■ Centrifuge at 3000 RCF for 5 min■ Store supernatant at 0-8 °C until
analysis
DBS Cards Drying
DBS Extraction
Instrumental Conditions (UPLC)
Example Chromatograms – Carryover
Calibrator PerformanceCurve Performance Over Three Core Accuracy and Precision Validation Runs
Results
Calibrator PerformanceIntra-Assay Accuracy and Precision of Quality Control Samples
Inter-Assay Accuracy and Precision of Quality Control Samples from ANOVA
Instrumental Conditions (MS)
The extraction of MX-01 from DBS was validated over the range of 1.00 to 1,000 ng/mL with a dilutional linearity limit of 40,000 ng/mL. Precision, accuracy, and selectivity met validation acceptance criteria. Storage stability was demonstrated for 201 d at room temperature (pending additional experiments), 6 days at -70°C, and 3 days at 45°C. Accurate quantitation was noted in both hemolyzed and hyperlipidemic samples. Furthermore, when the entire punch was excised and extracted during validation, MX-01 was accurately and precisely quantifi ed in spots prepared with hematocrit levels between 20 and 55%.
