Internal standard. Every bottle that you intend to quantitate carries the same known ISTD spike. The curve is built from ratios, so a weak injection moves both peaks together and still lands on the line.

ISTD initial calibration sequence Every bottle carries the same pink ISTD spike. Blue drops are the target. Curve uses ratios. ISTD  same spike in every bottle Pink drop = ISTD at dose IS. Blue drops = target. The curve uses response ratio vs concentration ratio. BLK ISTD only C1 low + IS C2 mid + IS C3 high + IS C4 highest + IS UNK-01 unknown + IS UNK-02 unknown + IS QC-01 check + IS ISTD amount is the same known spike in every vial (method dose IS). A missing ISTD peak flags the bottle. On the Quant, set ISTD to the ISTD analyte (Analyte|Quant when several QntKeys exist). ISTD analytes themselves fit average response factors. Per-sample ISTD concentration can override the method IS dose on that bottle.
Blank, multi-level cals, samples, and QC, each with a pink ISTD drop

When to use it

Set it up

  1. Create the ISTD analyte (type ISTD) and its Quant.
  2. Create the target analyte (type Target). On its Quant, set ISTD to that ISTD Quant. When several QntKeys exist, the cell shows Analyte|Quant.
  3. Keep the reserved dose name IS. Put the spike concentration on the ISTD analyte (and on the target if you override per bottle).
  4. Create C1…C5 (or your levels) for the target only. Those are the curve levels.
  5. Spike ISTD into every bottle you will read. Mark cals with Dose C1…C5. The ISTD amount stays IS.
  6. Analyze.

A blank in ISTD work usually still has the pink drop. It should show ISTD and no target.

How the curve is read

ISTD ratio calibration curve Y is analyte response divided by ISTD response. X is analyte concentration divided by ISTD concentration. ISTD curve  ratios, not raw areas A weak injection shrinks both peaks. The ratio stays on the curve. Analyte conc / ISTD conc Response / ISTD response UNK C1 C2 C3 C4 Each point is a pair X = C_analyte / C_ISTD Y = R_analyte / R_ISTD C_ISTD comes from dose IS or a per-sample override. If ISTD response is missing, the bottle cannot be read on an ISTD quant.
Ratio curve: analyte over ISTD on both axes
Axis Value
X Target concentration ÷ ISTD concentration
Y Target response ÷ ISTD response

Quanto resolves ISTD X and Y from the linked ISTD result in the same file (or the ISTD response stored on the calibration point). If ISTD response is missing or ≤ 0, that bottle cannot be read on this Quant.

ISTD analytes themselves are fitted as average of response factors. They are the denominator, not a second target curve.

Overrides

Mixing ESTD and ISTD

One method can have ESTD quants and ISTD quants. Key Review and reports still pick per analyte. A target without an ISTD link behaves as in ESTD.

Bracketing uses the same ratios; only the set of cal bottles changes. See Bracket calibration.