Example batches

These ten pesticide MRM batches show every calibration mode, both Cal group modes, and every curve fit, origin, and weight. One batch is an internal-standard calibration curve. One batch is spike recovery. One is standard addition. One is continuing calibration verification.

Download them as a ZIP:

Download the example batches (ZIP, 4.1 MB)

Unzip the folder, then open a file with File → Open batch…. The dialog title is Select Batch.

What you see when a batch opens

The original data files are not included. Sample paths look like Pesticides/Cal-L1.d. The chromatograms, peaks, results, and curves are already stored in the .qtb, so File → Open batch… shows them.

Analyze → Get signals reads those data files from disk. With this ZIP, leave Get signals alone and review the stored chromatograms in View → Samples and results → Result details.

Analyze → Quantitate fits the curves again from the stored peaks and responses. Linear curves with origin Force, and Average of response factors, show an R² in the file. Quantitate clears R² on those two fits. Other fits keep a normal R².

First order ln and Second order ln store Coeff 0 as the intercept and the next coefficients as the ln terms. That is the order the curve chart draws, so Quantitate leaves the line on the points. See Calibration curves and Quantitation.

What the batches share

Levels are 1, 2, 5, 10, 20, and 50 ng/mL, named L1 through L50. istd-calibration-curve.qtb adds L100 at 100 ng/mL. Units are ng/mL. Responses scatter by a few percent, and retention times drift slightly.

Most batches use these analytes:

curve-models.qtb also has Chlorpyrifos (13.40 min) and Lindane (7.35 min). istd-average-response-factors.qtb adds the internal standard Atrazine-d5 (8.38 min). istd-calibration-curve.qtb uses Atrazine, Simazine, Chlorpyrifos, and Diazinon, with Atrazine-d5 and Chlorpyrifos-d10 (13.32 min, 360.0 > 199).

A typical sample list is Solvent, Blank, Cal-L1 through Cal-L50, QC-low, QC-high, Creek-01, Apple-02, and Soil-03. Open it in View → Samples and results → Samples. The Type column is the role. CalGroup is the calibration group. Dose is the level name. See Batch and Doses.

Where a batch includes Metolachlor, Apple-02 Metolachlor has a qualifier ratio about 28% above the expected 62. Q1Act is the measured percent, Q1Rev is too high, Q fail is 1, and Q val is below the confirmation threshold. The qualifier uses its own Tolerance of 20 (general tolerance is off). See Qualifiers. Max QF is filled in on istd-average-response-factors.qtb, and that is where QF flag is too high.

Curve settings are on Method → Edit method… (F10), then View → Quants: columns Fit, Origin, and Weight. The calibration mode is Cal mode under Method → Properties… in that same editor. Cal group Mode is View → Cal groups in the method editor. The curve for the sample you are on is View → Samples and results → Calibration. See Bracketing and Charts.

Limit groups

Each sample has one LimitGroup. The catalog is Method → Edit method…, View → Limit groups. The numbers are View → Limits. See Limit groups and Checks.

The external batches use six groups:

istd-average-response-factors.qtb uses those six groups. Atrazine-d5 is spiked into every vial, so its row is concentration too high 30 ng/mL and recovery 70–130% in each group, including Blank.

istd-calibration-curve.qtb uses Blank, Calibrators, QC, and Samples. Samples covers Creek-01, Apple-02, Soil-03, and Matrix-01: concentration 0.2–50 ng/mL, recovery 70–130%. Atrazine-d5 and Chlorpyrifos-d10 are spiked into every vial, so each ISTD row is concentration too high 30 ng/mL and recovery 70–130% in every group, including Blank. That recovery window is the ISTD response window.

standard-addition.qtb uses Addition and Blank. Addition covers the five Sample aliquots: concentration too low 0.5 ng/mL, too high 50 ng/mL, recovery 70–130%, minimum R² 0.995. Blank is concentration too high 0.5 ng/mL.

ccv.qtb uses Calibrators, CCV, and Routine. Calibrators are the five calibration levels (too high 200 ng/mL, recovery 70–130%). CCV is the three verification vials, with recovery 80–120%. Routine covers QC-mid and Field-01 through Field-09: concentration 0.2–100 ng/mL, recovery 70–130%. MaxCcvGap is 3 and CloseCcv is on for Routine. See Continuing calibration verification.

Most results sit inside those windows. C flag, RecFlag, R² flag, and QF flag are on View → Samples and results → Results. The rows that fall outside are named on each batch below.

initial-external-linear.qtb

Initial calibration, external standard. Cal group Pesticides, Mode Calibration.

Atrazine is the usual new-quant default: a straight line through the origin. Simazine includes (0, 0) as a point. Diazinon weights by 1/y.

Look at: Samples for the full sequence and LimitGroup. Calibration on Atrazine (through the origin) and on Simazine (the origin point is on the line). Results: Apple-02 Atrazine is about 6.4 ng/mL, so C flag is too high against the Fruit limit of 5. QC-high Atrazine RecPct is 68, so RecFlag is too low against the QC window of 80–110. Creek-01 is about 0.45 ng/mL and stays above the Water too-low of 0.2. Apple-02 Metolachlor still shows Q1Rev.

Try this: In View → Quants, change Diazinon Weight from 1/y to Equal. Analyze → Quantitate. Open Calibration again and compare the line and R². Quantitate also clears R² on the Force lines.

partial-external-linear.qtb

Partial calibration. Every target is Linear, origin Force, weight Equal. Cal group Pesticides, Mode Calibration.

Cal-L2 is a stored calibration response. The sample list has the other five levels as live injections. QC-low uses dose L5.

Look at: Samples, then Calibration for any analyte. The curve still has an L2 point. Results: Creek-01 Atrazine is about 0.43 ng/mL, so C flag is too low against the Water reporting limit of 1 ng/mL. The other analytes on Creek-01 stay above their too-low of 0.2.

Try this: Analyze → Quantitate. L2 stays on the curve. Partial keeps a stored response when that dose has no live calibration injection. See Bracketing.

bracket.qtb

Bracket. Cal group Pesticides, Mode Calibration. Every target is Linear, origin Force, weight Equal.

The list is a morning block (AM-L1 through AM-L50), then Solvent, Blank, QC-low, Apple-02, and Creek-01, then an afternoon block (PM-L1 through PM-L50), then Soil-03.

Each analyte has three brackets. The morning block is its own curve. The middle samples use the morning block and the afternoon block together. Soil-03 uses the afternoon block. On that afternoon curve, PM-L50 has Use off, so that fit omits L50.

Look at: Samples for the two calibration blocks and LimitGroup. Select Soil-03, open Calibration, and find PM-L50 with Use off. Select Apple-02 and the curve includes both blocks, with PM-L50 still in the fit. Bkt on the result is the bracket index. Results: Blank Atrazine is 0.8 ng/mL, so C flag is too high against the Blank limit of 0.5. The other analytes on Blank stay under that limit.

Try this: On the Soil-03 curve, turn PM-L50 Use on. The line refits and R² changes. Turn it off again to put the example back.

bracket-with-borrow.qtb

Bracket with borrow. Same samples, analytes, and fits as bracket.qtb.

PM-L50 Use is off on the afternoon bracket. The afternoon curve fills that level from the morning AM-L50 response.

Look at: Select Soil-03 and open Calibration. PM-L50 is off, and the L50 response on that curve matches AM-L50. Select Apple-02 to see the combined middle curve. The six limit groups match bracket.qtb. Blank Atrazine is under the 0.5 ng/mL blank limit, and the results stay inside their windows.

Try this: Turn PM-L50 Use on. The afternoon curve then uses the afternoon injection. Turn it off again and the morning response is back on that level. See Bracketing.

istd-average-response-factors.qtb

Initial calibration with an internal standard. Cal group Pesticides, Mode Calibration.

Atrazine-d5 is type ISTD, fit Average of response factors, origin Force, weight Equal. Its dose is IS = 10. Atrazine, Simazine, and Metolachlor are Linear, origin Force, weight Equal. Their curve y is the area ratio (analyte area / Atrazine-d5 area).

Look at: Method → Edit method…, View → Quants. The ISTD column on the three targets is Atrazine-d5. In Results, Rsp on a target is the ratio and ISTD rsp is the Atrazine-d5 area. Calibration on Atrazine is a line through the origin in ratio units. Calibration on Atrazine-d5 is the average response factor. Apple-02 Metolachlor has Q fail 1 and Fruit Max QF 0, so QF flag is too high. Atrazine-d5 recovery stays near 100%, inside 70–130%, including on Blank.

Try this: Analyze → Quantitate. R² clears on Atrazine-d5 and on the target lines, because those fits are Average of response factors and Force. The concentrations stay. See Quantitation.

istd-calibration-curve.qtb

Initial calibration with an internal standard and a fitted curve. Cal group Pesticides, Mode Calibration.

Atrazine and Simazine use Atrazine-d5. Chlorpyrifos and Diazinon use Chlorpyrifos-d10. Both internal standards are type ISTD, fit Average of response factors, origin Force, weight Equal, dose IS = 10 ng/mL in every vial. The four targets are Linear, origin Ignore, weight 1/x.

The target curve is built in ratio units. Curve x is analyte concentration / ISTD concentration. Curve y is analyte area / ISTD area. Origin Ignore fits an intercept, so the line is y = a + b·x. Weight 1/x gives the low levels more say. To read a sample, Quanto divides the analyte area by that vial’s ISTD area, inverts the line, and multiplies by the ISTD concentration (10 ng/mL). A vial whose ISTD area is low still reports the analyte concentration from the ratio. The bracket stores the formula, R², and the relative residuals. A relative residual is (back-calculated concentration − known concentration) / known concentration, in percent. The largest residual on each target is about 1%.

The sequence is Blank, Cal-L1 through Cal-L50, Cal-L100, QC-mid (dose L10), Creek-01, Apple-02, Soil-03, and Matrix-01. ISTD areas move about ±2% from vial to vial. On Matrix-01 both ISTD areas are about 60% of the other vials. The analyte areas on that vial are lower by the same factor, so the ratio stays on the curve. Atrazine-d5 RecPct is 60 and Chlorpyrifos-d10 RecPct is 59. Both are under the 70–130% window, so RecFlag is too low. Target concentrations on Matrix-01 stay near 8 ng/mL, inside the Samples window of 0.2–50. QC-mid recovery stays near 100%, inside 80–110%. Blank target concentrations stay under 0.5 ng/mL.

Look at: Method → Edit method…, View → Quants. The ISTD column is Atrazine-d5 or Chlorpyrifos-d10. On a target, Fit is Linear, Origin is Ignore, and Weight is 1/x. Calibration on Atrazine shows the intercept, the formula, R², and the residuals. In Results, Rsp is the peak area and ISTD rsp is the internal-standard area. On Matrix-01, the ISTD area is about 60% of a calibration vial, RecPct is about 60, RecFlag is too low, and the target concentrations are still about 8 ng/mL.

Try this: Analyze → Quantitate. The linear Ignore curves keep R². Matrix-01 concentrations stay, because the area ratio is unchanged. See Quantitation and Checks.

curve-models.qtb

Initial calibration. Cal group Pesticides, Mode Calibration. Six analytes, and two injections at every level (Cal-L1 and Cal-L1-b, and the same for the other levels). The second injection is what 1/SD^2 needs.

Lindane’s intercept is the blank offset, 1500, from Blank with Blank use Average (CalGroup).

Look at: View → Quants for Fit, Origin, and Weight. Samples for the paired calibration injections. Calibration on each analyte: the quadratic bend on Atrazine, the power curve on Simazine, the segments on Metolachlor, the log curves on Diazinon and Chlorpyrifos, and the raised intercept on Lindane. The Diazinon and Chlorpyrifos lines follow their points. Soil-03 Chlorpyrifos R² is 0.994, just under the Soil minimum of 0.995, so R² flag is too low. Cal-L20 and Cal-L20-b on that analyte recover at about 116% and 87%, which is what pulls the R² down, and both stay inside the Calibrators window of 70–130%. The other groups leave minimum R² empty, so the same curve leaves their R² flag OK. Apple-02 Metolachlor still shows Q1Rev.

Try this: Select Soil-03 and read Chlorpyrifos R² and R² flag on Results. Analyze → Quantitate, then open Calibration on Diazinon and on Chlorpyrifos. The line stays on the points. On Atrazine, change Weight from 1/SD^2 to Equal, Quantitate, and compare the quadratic.

spike-recovery.qtb

Initial external curves, all four targets Linear, origin Force, weight Equal. Cal group Pesticides, Mode Calibration. Spike recovery sits on that curve. See Spike recovery.

Apple-unspiked is type Sample with an empty Dose. Apple-spike is type Sample, dose L20. QC-spike is type QC, dose L20. All three share SpikeGroup LotA. LotA is colour Pink on the method spike-group list. The Samples SpikeGroup cells for those three vials use that colour.

RecPct is about 106 on Apple-spike and about 93 on QC-spike. NConc is Conc minus the on-column dose (20 ng/mL). Apple-unspiked keeps its curve Conc and has an empty RecPct.

Look at: Samples, columns SpikeGroup (pink for LotA), Dose, LimitGroup, and Type. View → Spike groups shows LotA and its colour. Results for those three samples: Conc, NConc, and RecPct. Apple-spike is about 106% and QC-spike is about 93%, inside the Fruit window of 70–130% and the QC window of 80–110%. Creek-01 stays above the Water too-low of 0.2 ng/mL. Apple-02 is in this batch as well, with the Metolachlor qualifier flag. The calibration samples in the same CalGroup are the curve.

Try this: Clear SpikeGroup on Apple-spike and Analyze → Quantitate. RecPct becomes 100 × Conc / dose. Put LotA back and Quantitate to restore the spike recovery.

standard-addition.qtb

Cal group Addition, Mode Standard addition. One analyte, Atrazine, Linear, origin Ignore, weight Equal. The method Cal mode is Initial. The vials are Sample and Blank. See Standard addition.

The four points with Use on fit a straight line with R² above 0.995. NConc is the x-intercept (intercept / slope), about 1.94 ng/mL. Spiked RecPct is 100 × (Conc − NConc) / dose. Spike-10 is stored off the line (its response is far below the other points).

Look at: In the method editor, View → Cal groups, row Addition, Mode Standard addition. View → Limit groups shows Addition and Blank. Samples for CalGroup Addition, LimitGroup, and the doses. View → Samples and results → Calibration: the chart title is Standard addition · Addition, the x-axis is Added dose, and the intercept is marked NConc. Spike-10 has Use off and sits off the line. The addition line uses the four fitted points: Unspiked, Spike-1, Spike-2, and Spike-5. Blank is in Samples, concentration 0, under the blank limit of 0.5 ng/mL. The Addition R² is about 1.000, above the minimum of 0.995, and the spiked concentrations stay under the too-high limit of 50 ng/mL.

Try this: Turn Spike-10 Use on. The line refits and R² drops. Turn Use off to restore the example.

ccv.qtb

Initial calibration, external standard, every target Linear, origin Force, weight Equal. Cal group Pesticides. The sequence is Cal-L1, Cal-L5, Cal-L10, Cal-L20, Cal-L50, then CCV-1, Field-01, Field-02, QC-mid, Field-03, CCV-2, Field-04, Field-05, CCV-3, Field-06, Field-07, Field-08, and Field-09.

Each CCV uses dose L10 (10 ng/mL) and limit group CCV (recovery 80–120%). The curve fit uses the five calibration levels. CCV-1 and CCV-3 read 10 ng/mL, so RecPct is 100 and DevPct is 0. CCV-2 reads 6 ng/mL, so RecPct is 60, DevPct is −40, and RecFlag is too low.

MaxCcvGap is 3 and CloseCcv is on for Routine.

The calibration levels keep CcvFlag OK. Field concentrations are about 4 ng/mL, inside 0.2–100. QC-mid recovery is about 100%, inside 70–130%.

Look at: Samples for Type CCV and LimitGroup CCV. With Show sample type symbols on, the type column prefixes ▲. View → Limit groups for MaxCcvGap 3 and CloseCcv on Routine. View → Limits for the CCV recovery window 80–120. Results for RecPct, DevPct, RecFlag, and CcvFlag on Atrazine. Calibration: the five standards are the line, and the CCV vials are orange diamonds off that fit.

Try this: Set CCV-2’s response back to the L10 response and Analyze → Quantitate. Field-04 and Field-05 return to CcvFlag OK. Put a CCV after Field-09, or raise MaxCcvGap, and Quantitate again to clear the trailing interval flags.