Mass Spectrometry Quantitation

Standards and Standard Curve Preparation

for

Quantitative PK/MS Analysis

Introduction

The integrity of a quantitative assay depends on the quality of the reference standard. A true reference standard must pass a series of tests which make up the reference standard qualification protocol. The reference standard qualification tests are designed to fully characterized the reference standard as to it's quality and content. If the content of the reference standard is incorrect then any quantitative assay that is based on that standard will be incorrect. However, when performing "research PK" there may not be time to fully characterize the compound, for the simple fact that the lab may be evaluating three new compounds a week. When faced with developing a "research PK" quantitative assay the laboratory should make an effort to establish a pseudo reference standard that does not change during the evaluation of the compound and that can be permanently archived for future reference. This procedure ensures that every PK experiment can be measured relative to this pseudo reference standard. The standard curve creation procedure is also a critical part of the quantitative assay. In order for the assay to be valid the reference standard must be valid and each weighing and pipetting in the standard curve creation must be performed correctly. Below we describe how we prepare a typical standard curve for a quantitative PK/MS assay.

Preparation of the Standard Curve

If at all possible it is helpful to know the range of concentrations in the unknown samples before planning out the standard curve. A quick estimate can be made by shooting an early, middle and late time point along with a low, middle and high standard. This sneak peak will allow a more appropriate standard curve creation range and will tell you how much sample needs to be injected. This procedure may keep you from repeating a very lengthy assay. Some time points may contain high concentrations of the target compound. This preliminary analysis may allow you to dilute, for example, the earliest time point by 10X allowing you to build a more appropriate standard curve.

Example Standard Curve Dilution Scheme (your actual procedure may differ radically, use only as an example)

The dilution scheme shown in the table below describes a 3X, (3X50 µl), preparation and attempts to closely simulate the preparation of the unknown samples. The table includes the addition of the organic solvent for the plasma crash. The standard sample preparation and volume injected for analysis should mirror the unknown sample work-up and analysis.

Weigh out approximately 1 mg of reference standard and reconstitute to a concentration of 10 µg/ml. Weighing less than 1 mg may magnify the possibility of weighing and transfer errors. Certain hydrophobic compounds may require a two step reconstitution. For example, a hydrophobic compound may not be soluble in a high aqueous solution. Try dissolving the compound in a small amount of DMSO then attempt the second step of the reconstitution in the lowest organic content solution in which the compound is stable and soluble. The medicinal chemist that supplied the compound for evaluation can often advise you as to the solubility and stability characteristics of the compound.

(warning this dilution scheme may contain errors you must validate it for your own purposes)

Reference Standard Concentration = 10 µg/ml (Prepare in non-serum containing solution)

Sol A = 1 µg/ml (100 µl Ref. Std. + 900 µl Serum)

Sol B = 100 pg/ml (10 µl Ref. Std. + 990 µl Serum)

Sol C = 10 pg/ml (10 µl Ref. Std. + 9990 µl Serum)

Sol D = 1 pg/ml (10 µl Sol B + 990 µl Serum)

Level Conc. .
Standard
Solution
.
Serum

Internal
Standard
.. Organic

. .. . X +
X
30 µl + 420 µl

. . . . . . and mix . and mix

. . . . . . . . .

1 500 fg/ml . 75 µl Sol D + 75 µl 30 µl + 420 µl

2 1 pg/ml . 150 µl Sol D +
0 µl
30 µl + 420 µl

3 5 pg/ml . 75 µl Sol C + 75 µl 30 µl + 420 µl

4 10 pg/ml . 150 µl Sol C + 0 µl 30 µl + 420 µl

5 20 pg/ml . 30 µl Sol B + 120 µl 30 µl + 420 µl

6 50 pg/ml . 75 µl Sol B + 75 µl 30 µl + 420 µl

7 80 pg/ml . 120 µl Sol B + 30 µl 30 µl + 420 µl

8 100 pg/ml . 15 µl Sol A + 135 µl 30 µl + 420 µl

9 200 pg/ml . 30 µl Sol A + 120 µl 30 µl + 420 µl

10 500 pg/ml . 75 µl Sol A + 75 µl 30 µl + 420 µl

11 1000 pg/ml . 150 µl Sol A + 0 µl 30 µl + 420 µl

. . . . . . . . .

Download the standard curve method in Excel format by right clicking on the icon and choose "save target as" to save the file to your computer. (warning this dilution scheme may contain errors you must validate it for your own purposes)

Method Notes:

The internal standard should be thoroughly mixed in with the sample before adding the organic for the crash. If at all possible try to add the internal standard in a low organic solution so that the addition of the I.S. does not cause a premature precipitation. If possible an internal standard stock prepared in serum is best.
Solutions A, B, C and D should be prepared in serum so that the standard curve samples more closely resemble the unknown sample matrix.
Avoid serial dilutions when creating a standard curve. If a mistake is made in creating the first dilution that mistake will be propagated throughout the entire curve.
Try not to pipette less than 10 µl, smaller volumes may lead to greater errors. Ensure that your pipettes are calibrated.
Total volume of serum used in this procedure is approximately 12.7 ml

Running the Standard Curve

Since the standard curve can often span three orders of magnitude usually the standard samples are run from lowest concentration to highest. This procedure guards against high level standard chromatographic carry-over. After the standard curve is run, several blank samples should be run until no carry-over is noted. Likewise, sample sets are often run in reverse order with the latest time point run first and the earliest time point run last. Early time points can have the highest level of compound and thus present the highest risk of carry-over. Each PK time course should be separated by several blank aqueous solvent injections to guard against carry-over.

Randomizing Standards and Samples

Some labs have a method for randomizing samples before they are run. This procedure ensures that no sample is unfairly biased. The randomization procedure is often used with a chromatographic method that has been fully characterized and validated. If you follow the randomization procedure you must be certain that you have a robust chromatographic method that does not provide significant carry-over. Randomization suitability should be validated into the method if it is to be used.

Running System Suitability

System suitability is one of the most important components of any analytical assay. System suitability is required to be passed before the samples can be run. System suitability provides reasonable assurance that the; HPLC, column, and mass spectrometer are performing adequately before launching into a large sample set. While it is true that your system can die at any point during the assay even if you pass your initial specification, YOU SHOULD NEVER LAUNCH INTO AN ASSAY WITHOUT PASSING SYSTEM SUITABILITY! A system suitability test answers the question, "Is my assay working? and Should I proceed with sample analysis?" System suitability is different from lab to lab and assay to assay. In our lab, system suitability for a quantitative assay includes: chromatographic peak shape metrics, passing a defined allowable limit of detection, running the full standard curve and passing certain metrics for linearity and % RSD and finally passing the blank run tests before launching into the sample set. Since columns can die in the middle of a large sample set we have made a rule that we retire all columns after 500 plasma crash injections. Large assays are costly and time consuming, discarding the column ensures that less time and money will be wasted. I know, it is difficult to see a good HPLC column retired but in the end less time will be spent in the lab trouble shooting an assay that has stopped working.

The "QCs"

The QCs are a set of standards used to check the accuracy of the standard curve described above. The QCs are normally set to fall in the low, middle and high regions of the standard curve and are run interspersed with the unknown samples. One can think of the QC as an independently weighed reference standard. In the strictest sense a second analyst would weigh out the reference standard for the QC on a separate balance. This separate standard, the QC, controls for operator and balance bias and provides a necessary double check of the fitness of the official standard curve. The evaluation of the QC samples have a large impact on whether a particular assay was valid or not. It is wise to build an evaluation of the QC samples into the initial system suitability test as well as having them interspersed with the unknown samples.

THE END

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