Validation

The performance of a kit run is assessed here.

To validate a plate run:

• Uploaded data, see Upload MS data
• Use data available in the database, see Load MS data from database

To protect validation statuses and results from alterations, use Approve plate run.

Validate plate run

To assess the performance of a kit run, click Validate.

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Wait until the validation is completed.

The progress is indicated as a blue bar at the upper left edge of the screen.

Validation features

Within the validation section, the following features are available.

Summary

• Plate run: Shows plate run barcode. Click to load the corresponding data directly in Results.
• Run time: date of MS data uploaded
• Plate validation: "OK" or "Not OK"

  OK: Plate successfully passed the validation criteria.

  Not OK: Plate did not fully meet the validation criteria. Please review "Analyte results" window.

• Note: Click to open a summary of all validation notes, including a summary of excluded metabolites or wells.

LOD algorithm

The limit of detection (LOD) for each metabolite within a kit run is calculated from the concentrations in the zero samples based on either the standard deviation (StdDev-based) or median (Median-based).

• StdDev-based: LOD is calculated as median concentration of the zero samples concentrations plus three times the standard deviation of the zero samples concentrations.
  LOD = median background signal + 3 × standard deviation
• Median-based: LOD is calculated as three times the median concentration of the zero samples.
  LOD = median background signal × 3

Requirement for StdDev-based algorithm:

At least two replicates of a zero sample of the same material per plate run, e.g. two PBS replicates.

If StdDev-based or Median-based are defined as default in the Settings > LOD algorithm, this overrules the predefined LOD algorithm of the method (OP).

Default LOD algorithm

The default LOD algorithm to use for every kit can be defined in the Settings, refer to section LOD algorithm.

Method default: LOD is calculated according to default settings in the method's OP, "StdDev-based" or "Median-based".

StdDev-based: this algorithm is always used.

Median-based: this algorithm is always used.

For validated plate runs, the used LOD algorithm is shown, e.g. "Median-based".

When repeating the Validation of a validated plate run and the default LOD algorithm is different from the one previously used, a warning is shown.

Example

• Current LOD algorithm of plate run is "Median-based"
• Default LOD algorithm is "StdDev-based"
• Warning is shown if current (= "Median-based") or new (= "StdDev-based") LOD algorithm is used for plate run validation.
  

LOQ definition

The limit of quantification (LOQ) is defined as

LOQ = LOD × 3.3 .

Link sample material with zero samples

If zero samples of different materials are available on one plate run, sample materials need to be linked. Please find an example below.

Zero samples

• PBS
• Isoprop_H2O 80_20

Sample materials

• EDTA plasma
• liver tissue

EDTA plasma is linked with PBS by dragging EDTA plasma to PBS.

liver tissue is linked with Isoprop_H2O 80_20 by dragging liver tissue to Isoprop_H2O 80_20.

This definitions are used for limit of detection (LOD) calculations.

• The zero sample PBS is used for LOD calculations of EDTA plasma samples.
• The zero sample Isoprop_H2O 80_20 is used for LOD calculations of liver tissue samples.

  

Plate view

To assess the performance of a plate run, select a QC sample from the Plate view.

The size and level of detail in the plate view is adjustable.

small size

sample IDs shown

sample barcodes shown

large size

1. Select a sample, e.g. QC2 on position E2, by clicking on the corresponding well.

   

2. Validation results are displayed in the tables Analyte results and ISTD results.

   

   CVs are calculated, when at least three QC well replicates are available per plate run.
   CV = standard deviation / mean concentration * 100

   Accuracies are calculated for quantitative metabolites only, analyte classification T1.

   • Accuracies are calculated for metabolites of analyte classification "T1"
   • CVs are calculated for metabolites of analyte classification "T1" or "T2"

3. Validation results are illustrated in the graphics Analyte results and ISTD results.

   

   CVs are shown as .

   Accuracies are shown as .

   Acceptance ranges are shown as in the corresponding color of or .

   

   ISTD intensities are shown as

   Acceptance ranges are shown as

4. Concentrations of samples and counts of internal standard (ISTD) are validated based on acceptance range criteria, resulting in defined validation statuses.

• QC status: and are available.
• Calibration standard status: is available.
• 7-point calibrated metabolites quantification range: LLOQ and ULOQ are the lowest and highest enabled calibration standard level.
  Calibration standards can be enabled and disabled, see Disable calibration standard level
  Example: Cal2 - Cal7 are enabled. LLOQ = concentration of Cal2, ULOQ = concentration of Cal7.

Chromatograms

Chromatograms to a corresponding well can be displayed.

• Click on one well, e.g. QC2 on position E2.
• Select an analyte, e.g. Ala.

Analyte classification

• Results from all biocrates kits are quantitative, default unit is µmol/L.
• All results are precise and highly reproducible, showing low variations (CVs).
• Defined metabolites use an external seven-point calibration. The calibration is performed in Quantification > Calibration.
• Further metabolites (non-seven-point calibrated) use an internal one-point calibration. For quantification an internal standard (ISTD) is used.
• For details, refer to the kit specific documents "Analytical specifications".

Analyte classification	Information
Type 1 (T1)	Accuracy checked during kit validation. Reproducible (precise) results with low CVs and good accuracy (accurate).
Type 2 (T2)	Reproducible (precise) results with low CVs.
Not validated (NV)	Validation not possible, e.g. metabolite not available in validation matrix.

Validation statuses

• All validation statuses like <LOD, <LLOQ, or >ULOQ refer to the measured concentrations.
• "Measured concentrations" are values, to which no calculations were applied, e.g. data normalization or sample volume different from 10 µL.
• When applying any calculation or normalization to results, concentrations will change but the validation status is kept.
• Sample material specific LOD: When using zero samples of different materials, e.g. "serum" and "tissue", material specific LOD values are used, according to section Link sample material with zero samples.
• Summary: Validation statuses like <LOD, <LLOQ, or >ULOQ are given before calculation or normalization procedures are performed.

Example

• Check for each measured concentration: above or below the LOD.

• Measured concentration below the LOD receive the status .

• Calculation or normalization procedures may change the concentration shown e.g. in Results, but not the validation status.

• Summary: After calculation or normalization procedures were performed, concentrations may be flagged as but are shown above the LOD.

  Values in bold: concentration and status do not match. After normalization, concentration of sample A is shown above the LOD but the status remains .

  	Sample A	Status	Sample B	Status
  Measured concentration	0.122		0.198
  Normalized concentration	0.433		0.322

  LOD: 0.150

  LLOQ: 0.200

  Unit: µmol/L

Analyte results

Validation status of concentration.

Available for sample types calibration standard, zero, QC, unknown.

concentration is within the quantification range, see kit specific document "Analytical specifications"

concentration is below the limit of detection (LOD)

concentration is below the defined "height threshold", FIA data only

concentration is below the lower limit of quantification (LLOQ), but above the LOD

concentration is above the upper limit of quantification (ULOQ)

CV is outside the acceptance range

accuracy is outside the acceptance range

intensity of the internal standard (ISTD) is outside the acceptance range

ISTD results

Validation status of internal standard intensity.

Available for all sample types.

intensity is within the acceptance range

intensity of the internal standard (ISTD) is outside the acceptance range

Blank

ISTD signals (MRMs) are checked exclusively.

below upper intensity (cps) acceptance limit

if above intensity (cps) acceptance limit

Quality assessment

For an easy quality assessment of a kit run, validation statuses are given on metabolite, well/injection, and plate run level.

• Quality assessment performed is sample type and analyte classification (T1 or T2) specific.
• "Excluded" metabolites, injections, or wells are not used for quality assessment.

Metabolite level: a calculated concentration for a metabolite is evaluated.

Injection level: all concentrations of an injection are evaluated.

Well level: all injections from one well are evaluated. If one injection per well was performed, injection level = well level.

Plate run level: all wells of one plate run are evaluated.

Custom QC: "QC" and "Custom QC" samples are grouped under the term "QC".

Validation criteria are only given if not described in section Validation statuses.

Metabolite level

Calibration standard, acceptance criteria

• accuracy within kit acceptance criteria, in general 70-130%
• accuracy not within kit acceptance criteria, in general 70-130%

QC, acceptance criteria

• Accuracy calculated exclusively for metabolites of analyte classification "T1".
• CV calculated exclusively for metabolites of QCs and of analyte classification "T1" or "T2".
  Requirement: at least three QC replicates per plate run and concentration above LOQ.

• accuracy within kit acceptance criteria, in general 70-130%, and CV below 20% (analyte classification T1) or below 30% (analyte classification T2)
• accuracy not within kit acceptance criteria, in general 70-130%
• CV above 20% (analyte classification T1) or above 30% (analyte classification T2)

Well/injection level

Well/injection status is , if

at least 85% metabolites per well/injection with an analyte classification T1 are and

at least 80% metabolites per well/injection with an analyte classification T2 are .

For this evaluation, Valid means for

• metabolites T1 not Accuracy ot ouf range or CV out of range,
• metabolites T2 not CV out of range.

Plate run level

Plate run validation requirement: at least three QC replicates of one level per plate run.

For plate run validation, QCs are exclusively used.

• OK if at least 67% of all wells per plate run have a well status
• Not OK if less than 67% of all wells per plate run have a well status

Well status Valid means not CV out of range or Accuracy out of range of QC samples, as described in Well/injection level.

Exclude metabolite, well, sample type, or plate run

A metabolite, sample, well, or plate run can be excluded. This may be required, e.g. when a sample was pipetted incorrectly onto one well position or the incorrect retention time (RT) was defined for one metabolite in the corresponding acquisition method.

Exclude a metabolite

1. Select a well, e.g. QC2 on position E2.

   

2. Right click on a metabolite, e.g. Ala.

   

3. Add a note, e.g. "Incorrect RT defined" and click Save.

   

Multiple metabolites can be selected. Click in the field Metabolite, make a selection, and click Save.

Exclude a well

1. Right click on a well, e.g. QC2 on position E2.

   

2. Add a note, e.g. "Incorrect pipetting" and click Save.

   

• Multiple wells can be selected. Click in the field Well, make a selection, and click Save.

  

• Samples from the same type, e.g. all QCs, are selected by selecting QCs.

  

Exclude a plate run

1. Above the plate, click Exclude.

   

2. Add a note, e.g. "Kit run failed" and click Save.

   

Add notes

Notes can be added on metabolite, well, or plate run level.

1. Select a metabolite or well and right click, e.g. QC2 on position E2.

   

2. Add a note and click Save.

   

• Plate notes can be added, by right click on .

  

Multiple metabolites or wells can be selected. Click in the field Metabolite or Well, make a selection, and click Save.

Approve plate run

Approve plate run requires WebIDQ core+.

• To protect validation statuses and results from alterations click Approve plate run.
  
  WebIDQ functionalities which would result in integration, validation status, or results alterations are now disabled.
• To allow alterations of a plate run, click Unapprove.
  

Enable or disable the functionality approve plate run in the settings.

This is a global setting, affecting all users.

Disabling the setting "Approve plate runs" resets all approved plate runs to unapproved!

Changing the option "Approve plate runs" in the settings requires the WebIDQ user role admin.

Change WebIDQ roles in mybiocrates > users, see mybiocrates manual section Edit users.