Article

pubs.acs.org/ac

Selecting the Correct Weighting Factors for Linear and Quadratic

Calibration Curves with Least-Squares Regression Algorithm in
Bioanalytical LC-MS/MS Assays and Impacts of Using Incorrect
Weighting Factors on Curve Stability, Data Quality, and Assay
Performance
Huidong Gu,* Guowen Liu, Jian Wang, Anne-Françoise Aubry, and Mark E. Arnold
Bioanalytical Sciences, Research & Development, Bristol-Myers Squibb Company, Route 206 & Province Line Road, Princeton, New
Jersey 08543, United States
*
S Supporting Information

ABSTRACT: A simple procedure for selecting the correct

weighting factors for linear and quadratic calibration curves with
least-squares regression algorithm in bioanalytical LC-MS/MS assays
is reported. The correct weighting factor is determined by the
relationship between the standard deviation of instrument responses
(σ) and the concentrations (x). The weighting factor of 1, 1/x, or
1/x2 should be selected if, over the entire concentration range, σ is a
constant, σ2 is proportional to x, or σ is proportional to x,
respectively. For the first time, we demonstrated with detailed
scientific reasoning, solid historical data, and convincing justification
that 1/x2 should always be used as the weighting factor for all
bioanalytical LC-MS/MS assays. The impacts of using incorrect
weighting factors on curve stability, data quality, and assay
performance were thoroughly investigated. It was found that the most stable curve could be obtained when the correct
weighting factor was used, whereas other curves using incorrect weighting factors were unstable. It was also found that there was
a very insignificant impact on the concentrations reported with calibration curves using incorrect weighting factors as the
concentrations were always reported with the passing curves which actually overlapped with or were very close to the curves
using the correct weighting factor. However, the use of incorrect weighting factors did impact the assay performance significantly.
Finally, the difference between the weighting factors of 1/x2 and 1/y2 was discussed. All of the findings can be generalized and
applied into other quantitative analysis techniques using calibration curves with weighted least-squares regression algorithm.

T he importance of establishing an accurate calibration

curve in quantitative analysis has been addressed by many
research groups.1−5 A well-established and interpreted
calibration curve is essential for the assay performance and
data quality,1 especially for bioanalytical liquid chromatog-
raphy-tandem mass spectrometry (LC-MS/MS) assays used in
drug development, where the FDA guidance6 and EMA
guideline7 on “Bioanalytical Method Validation” must be
strictly followed. The FDA guidance states very clearly
regarding the establishment of a calibration curve as the
following: “The simplest model that adequately describes the
concentration-response relationship should be used and
selection of weighting and use of a complex regression equation
should be justified”. Furthermore, selection of a regression
model correctly during the method development and validation
is critical for the smooth assay transfer from method validation
to sample analysis, from analyst to analyst, and from laboratory
to laboratory.
Two most commonly used regression models for LC-MS/
MS calibration curves are linear and quadratic regressions using
nonweighted or weighted least-squares regression algorithm.
Although the least-squares regression and curve weighting are
well-established statistics techniques, the “Test and Fit” strategy
is still widely used for selection of calibration curves and their
weighting factors in the bioanalytical LC-MS/MS community
because of its simplicity and lack of statistics data for the
regression model and weighting selection. With the “Test and
Fit” strategy, an incorrect weighting factor can be easily selected
because this strategy is based on the analyst’s subjective
interpretation of a limited set of test results. Besides the “Test
and Fit” strategy, several methods for selecting regression
model have been reported. Kiser and Dolan proposed a
systemic way to test and identify the best fit curve.8 By applying
regression with different weighting factor (1/x0, 1/x0.5, 1/x,
1/x2, and 1/x3) on a set of STD data, the suitable weighting
Received: February 17, 2014
Accepted: August 26, 2014
Published: August 26, 2014

© 2014 American Chemical Society 8959 dx.doi.org/10.1021/ac5018265 | Anal. Chem. 2014, 86, 8959−8966
Analytical Chemistry Article

scheme could be easily identified by choosing the one

generating the smallest sum of the relative errors. The whole
process can be programmed in a spreadsheet and takes only
seconds for the selection. However, this simply represents an
automated version of the “Test and Fit” approach. Almeida et
al.1 suggested that a weighting should only be used if
homoscedasticity was not met for the analytical data. Recently,
Steliopoulos et al.9 used several different functions to model the
dispersion of measurements with the concentration and then to
estimate the weights needed for the regression. By using Monte
Carlo calculations, Tellinghuisen investigated the effect of
improper weighting on linear calibration models.2 It was found
that for analytical data with heteroscedastic proportional error,
neglecting the weightings could result in as high as 1 order of
magnitude of precision loss in the low concentration region.
Another significant finding from this study was that the fitness
of calibration curve should not be used as the criteria in
selecting regression model and weighting factor because of the
“self-fulfilling” nature of the weighted least-squares regression
algorithm. Many research works recommended that 1/x2
weighting should be used for bioanalytical LC-MS/MS assays;
however, the recommendations were largely based on the “Test
and Fit” strategy.10−12 More recently, Hoffman introduced a
quantitative procedure for selecting weighting factors in
bioanalytical LC-MS/MS assays without using “Test and
Fit”.13 It was mentioned that this procedure had been adopted
by some bioanalytical laboratories in selecting weighting factors
for many years.
In this paper, we examined the justifications for using a Figure 1. (a) Instrument responses of 16 STD curves in eight
weighting factor based on the experimental data and proposed a analytical runs for BMS-708163 in human plasma LC-MS/MS assay
simple procedure for selecting the correct weighting factors for validation.16 (b) Standard deviation (σ) and variance (σ2) for
linear and quadratic calibration curves with least-squares instrument responses of 16 STD curves in eight analytical runs for
regression algorithm in bioanalytical LC-MS/MS assays. BMS-708163 in human plasma LC-MS/MS assay validation (STD
Using this simple procedure on historical data, we demon- curve range: 0.1−100 ng/mL).16
strated that 1/x2 should be the only right weighting factor for
LC-MS/MS assays. The impacts of using incorrect weighting deviations (σ, blue line) and variance (σ2, red line) for
factors on the curve stability, data quality, and assay instrument responses at each concentration level are shown in
performance were investigated. The difference between the Figure 1b. It was found that, from 0.1 to 100 ng/mL, σ
weighting of 1/x2 and 1/y2 was also discussed. Several critical increased in an approximately concentration-proportional
findings from this investigation can be generalized and applied manner, and σ2 increased in a much more than concentration
in other quantitative analysis techniques using calibration proportional manner. To address the heteroscedatic errors, a
curves with least-squares regression algorithm.

■
RESULTS AND DISCUSSION
The assumptions for the nonweighted linear and quadratic
regressions in bioanalytical LC-MS/MS assays are that the
errors from the dependent variable (y, instrument response) are
independent from each other (random), uncorrelated with the
independent variable (x, concentration), and have equal
variance (σ2) or standard deviation (σ) across the different
concentration levels (homoscedasticity14). However, a quick
review of any set of real life standard (STD) or quality control
(QC) data from assay validations or sample analysis will reveal
that the instrument response errors at different concentration
levels are actually correlated with the independent variable
(concentration) and have a larger variance (σ2) or standard
deviation (σ) at higher concentrations as long as the sample
size at each concentration level is large enough to provide a
rough estimate of variance. This is a clear indication that the
instrument responses have heteroscedatic error.15 Figure 1a
shows the instrument responses of 16 calibration curves in
eight analytical runs for a test compound (BMS-708163) in
human plasma LC-MS/MS assay validation.16 The standard
8960
modified least-squares algorithm, weighted least-squares, is
warranted. The idea is to assign an appropriate weight at each
concentration level which reflects the different measurement
uncertainties at different concentration levels.17 When the
measurements were uncorrelated and had different uncertain-
ties, Aitken demonstrated that the best unbiased estimates
could be obtained if each weight used was equal to the
reciprocal of the variance of the measurement.18,19 This is easy
to understand because the importance of each squared residual
at all concentration levels are normalized/equalized with the
reciprocal of the variance at the corresponding concentration
level.18,20,21 (Please see Supporting Information A for more
detailed discussion on linear and quadratic regression with
least-squares algorithm.)
Weight Estimation for LC-MS/MS Bioanalytical Assay
Calibration Curves Using Linear or Quadratic Regres-
sion with Weighted Least-Squares Algorithm. For the
estimation of weights for calibration curves in LC-MS/MS
bioanalysis, it is impractical to directly use 1/σi2 at each
concentration level for each individual run because the data set
is normally too small to estimate the σi, and the weights could
vary from run to run. The most common practice is to find an
dx.doi.org/10.1021/ac5018265 | Anal. Chem. 2014, 86, 8959−8966
Analytical Chemistry Article

Table 1. Theoretical Data for x and σ To Justify the Selection of 1, 1/x, or 1/x2 as the Weighting Factor
weighting factor concn x (arbitrary units) 1 2.5 5 10 50 100 250 350 500 RSD%
1 σ/x0 0.5000 0.5000 0.5000 0.5000 0.5000 0.5000 0.5000 0.5000 0.5000 0
σ/x0.5 0.0050 0.0079 0.0112 0.0158 0.0354 0.0500 0.0791 0.0935 0.1118 88.8
σ/x1 0.5000 0.2000 0.1000 0.0500 0.0100 0.0050 0.0020 0.0014 0.0010 171.1
1/x σ/x0 0.0354 0.0559 0.0791 0.1118 0.2500 0.3536 0.5590 0.6614 0.7906 88.8
σ/x0.5 0.0354 0.0354 0.0354 0.0354 0.0354 0.0354 0.0354 0.0354 0.0354 0
σ/x1 0.0354 0.0224 0.0158 0.0112 0.0050 0.0035 0.0022 0.0019 0.0016 106.0
1/x2 σ/x0 0.0050 0.0125 0.0250 0.0500 0.2500 0.5000 1.2500 1.7500 2.5000 130.1
σ/x0.5 0.0500 0.0316 0.0224 0.0158 0.0071 0.0050 0.0032 0.0027 0.0022 106.0
σ/x1 0.0050 0.0050 0.0050 0.0050 0.0050 0.0050 0.0050 0.0050 0.0050 0

empirical function between weights (1/σ2) and x or y to
approximate the variance with x or y.22,23 Fortunately, the
empirical function is likely predictable for data arising from
these assays and normally can be expressed in a simple power
relationship as σ = cyn (or σ = cxn, where c and n are
constants).24,25 Therefore, the weighting factor of 1/y2n (or
1/x2n) can be used as the weighting factor. As 1, 1/x, and 1/x2
are the most commonly used empirical functions for weighting
factor in LC-MS/MS assays, they were used in the initial
investigation, and the use of 1/y and 1/y2 were discussed in the
last part of this paper.
Selection of Weighting Factor Using Plots of σ ∼ x
and σ2 ∼ xA Qualitative Approach. The theoretical
relationships between σ and x, and between σ2 and x to justify
the selection of 1, 1/x, and 1/x2 weighting factors are shown in
Supporting Information B, Figure SI 1a−1c, respectively, and
the corresponding data are listed in Table 1.18,19 As shown in
Figure SI 1a−1c, the weighting factor of 1 should be used if a
linear relationship between σ and x0 is observed (σ and σ2 are
constants). The weighting factor of 1/x should be used if a
linear relationship between σ and x0.5 (or between σ2 and x1)
and a rightward quadratic relationship between σ and x1 are
observed. Similarly, the weighting factor of 1/x2 should be used
if a linear relationship between σ and x1 and a concave upward
quadratic relationship between σ2 and x are observed.
In regulated bioanalytical LC-MS/MS analysis supporting
nonclinical and clinical studies, a method has to be validated
before it can be used in sample analysis. A full method
validation consists of at least three accuracy and precision runs,
which can provide sufficient instrument response data from
STD or QC samples for the calculation of σ. Figure 1b and
Figure 2a−c16,26,27 show the relationships between σ and x
(blue line), and between σ2 and x (red line) from STD curve
instrument responses from four assay validations. A quick
eyeball of these plots suggested that 1/x2 should be used as the
weighting factor for 3 out of 4 assays listed, as very good
straight line relationships between σ and x and concave upward
quadratic relationships between σ2 and x were found over their
corresponding STD ranges. For the last assay, in most of the
STD curve range (0.1 to 80 ng/mL), the concave upward
quadratic curvature between σ2 and x was not obvious, and it
looked like that the relationships between σ and x, and between
σ2 and x followed the same linear trend. Actually, this was due
to σ being very close to 1 at 80 ng/mL so that the difference
between σ and σ2 over the concentration range of 0.1 to 80 ng/
mL was small and hard to discern graphically. However, over
the entire STD curve range, better linearity was found for the
relationship between σ and x. Therefore, 1/x2 should also be
used as the weighting factor for that assay.
8961
Selection of Weighting Factor Using Linearity
IndicatorsA Quantitative Approach. In some cases,
such as the last assay discussed above, the selection of a
relationship with better linearity may not be obvious using the
plots. An alternative, less empirical approach using three
linearity indicators is proposed for the selection of weighting
factor. The linearity indicators were defined as the relative
standard deviation (RSD%) of σ/x0, RSD% of σ/x0.5, and RSD
% of σ/x1, respectively. It is easy to see that, to justify the use of
1, 1/x or 1/x2 as the weighting factor, the RSD% of σ/x0, σ/x0.5,
or σ/x1 should be 0% as an exact linear relationship exists
between σ and x0, σ and x0.5, or σ and x, respectively, as shown
in Table 1. Therefore, in evaluating a real STD or QC data set,
the best linear relationship can be assumed to be the one that
gives the smallest linearity indicator. The linearity indicators
were calculated for all of the four assays discussed above. The
results (Table 2) confirmed the selection of 1/x2 as the
weighting factor for all of the four assays.
More than 20 LC-MS/MS assays developed in our
laboratories over a period of 6 years have been surveyed
using both the qualitative and quantitative approaches. The
assays covered small molecule and protein analysis with
different types of sample preparation and extraction techniques,
using different MS platforms for detection. The survey result
showed that 1/x2 was the only correct weighting factor for all of
the assays without any exception. This result is in agreement
with most bioanalysts’ “experience” that 1/x2 is the most
commonly used weighting, which have probably been built up
using the “Test and Fit” technique based on the overall
accuracy of the STD or/and QC data. The selection of 1/x2 for
all of the assays was also confirmed by weighted residual plots
as the weighted residuals are randomly distributed over the
entire concentration range.23 Another study we conducted on
uncertainty from LC-MS/MS measurement by calculating the
propagation of uncertainties from preparation of STD samples
to MS detection showed that a linear relationship actually
existed between σ and x, and therefore, the weighting factor
1/x2 should be used for all of LC-MS/MS assays (to be
published elsewhere).28 Any issues for bioanalytical assays (e.g.,
nonspecific adsorption, cross contamination, systematic bias,
failed QC due to preparation/storage/matrix difference
between STD and QC samples, etc.) may obscure this fact.
However, people should not manipulate the weighting factor to
deal with these issues for the sake of passing an analytical run.
All these issues should be resolved during assay development.
Impacts of Using Incorrect Weighting Factors on
Curve Stability, Data Quality, and Assay Performance. In
most cases, 1/x weighting and in some rare cases, even no
weighting, can also generate very good STD curves and QC
data, although the correct weighting factor should be 1/x2.
dx.doi.org/10.1021/ac5018265 | Anal. Chem. 2014, 86, 8959−8966
Analytical Chemistry Article

Figure 2. (a) Standard deviation (σ) and variance (σ2) for instrument responses of 10 STD curves in five analytical runs for BMS-512148 in dog
plasma LC-MS/MS assay validation (STD curve range: 50−2000 ng/mL). (b) Standard deviation (σ) and variance (σ2) for instrument responses of
14 STD curves in seven analytical runs for a PEGylated adnectin protein in monkey plasma LC-MS/MS assay validation (STD curve range: 50−25
000 ng/mL).27 (c) Standard deviation (σ) and variance (σ2) for instrument responses of eight STD curves in four analytical runs for BMS-708163 in
human CSF LC-MS/MS assay validation (STD curve range: 0.1−100 ng/mL).16

Here we show an example with STD data (Table 3a) from
BMS-708163 in mouse plasma validation with three accuracy
and precision runs, and the correct weighting factor is 1/x2.
Table 3b shows the linear regression results with weighting
8962
factors of 1, 1/x, and 1/x2 for each run using the original data in
Table 3a. Table 3c shows the regression results using the
original data with a couple of assumed instrument response
biases to test the curve stability. The quantitative comparisons
dx.doi.org/10.1021/ac5018265 | Anal. Chem. 2014, 86, 8959−8966
Analytical Chemistry Article

Table 2. Results of Linearity Indicator (RSD% of σ, σ/x0.5,
and σ/x) and Selection of Weighting Factor
weighting
RSD% RSD% RSD%
assay of σ/x0 of σ/x0.5 of σ/x1
Figure 1b 141.2 108.4 19.1
Figure 2a 104.2 60.2 7.5
Figure 2b 146.6 104.3 26.1
Figure 2c (STD curve range: 144.7 104.9 26.3
0.1 to 100 ng/mL)
Figure 2c (STD curve range: 147.5 98.8 28.0
0.1 to 80 ng/mL)
of curve stability for run 1 and 3 are shown in Table 3d.
Regulatory guidelines (e.g., FDA and EMA bioanalytical
guidance) were followed to discuss the data quality, assay
performance, and set the assay acceptance criteria. For non-
LLOQ STD and QC samples, ≤ ± 15.0% was used. For LLOQ
STD and QC samples, ≤ ± 20.0% was used.6,7
Curve Stability. Curve stability was defined as the resistance
of a STD curve against significant errors from one or more
STD samples. As the correct weighting factor for this assay was
1/x2, the influences of the errors at different concentration
levels on the calculation of the best fitting curve were not taken
into account at all when no weighting was applied. Therefore,
the STD curves were extremely unstable, and a small bias at the
high end of the STD curves could change the STD curves
significantly, especially at the low end of the STD curves, as
shown in Tables 3b, 3c, and 3d (run 3 with no weighting). By
using 1/x as the weighting factor for the assay, the influences of
the errors at different concentration levels on the calculation of
the best fitting curve were taken into account to some degree,
but it was still not enough to equalize the importance of the
errors at different concentration levels. As a result, the
instability of the curve could only be observed clearly when
there were significant biases at the high end of the STD curve,
as shown in Table 3b, 3c (run 1 and 2 with 1/x weighting) and
3d (run 1 with 1/x weighting). When the correct weighting
factor 1/x2 was used, the most stable curves were generated.
Any big biases from one or a couple of STD samples at any
concentration levels affected only those STD samples with big
biases, and had no or very little impact on the curves, as shown
in Table 3b and 3c (run 1 and 2 with 1/x2 weighting) and 3d
(run 1 with 1/x2 weighting). This can be easily understood as a
correct STD curve is “voted” equally by all of the STD samples
in a run (16 STD samples in our examples) when a correct
weighting factor is used. Therefore, it should not be sensitive
(or it should be stable) to any biases from only one or a couple
of STD samples. For example, as shown in Tables 3b−3d, when
the correct weighting factor of 1/x2 was used for run 1, only
−0.8% (at the STD curve low end) and 1.7% (at the STD curve
high end) predicted concentration changes were observed with
a −18.4% bias from one original instrument response. The only
consequence was that the STD sample with −18.4% bias
became an outlier (−18.8% deviation) and had to be excluded
from the STD curve, and this was exactly what should happen.
factor However, when the weighting factor of 1/x was used, the same
selected instrument bias caused −15.0% (at the STD curve low end)
1/x2 and 4.0% (at the STD curve high end) changes in the predicted
1/x2 concentrations. Therefore, the low STD sample with −23.2%
1/x2 deviation had to be excluded and the regression had to be
1/x2 performed again. As a result, an obvious instrument response
bias at the STD high end failed a total of six STD samples at the
1/x2 STD low end one by one, and eventually failed the run. More
substantial curve change can be observed with only −1.5% bias
from one original instrument response for a curve without
weighting when 1/x2 weighting is actually needed, as shown in
Tables 3b−3d, run 3.
It should be pointed out that different spacing of the STD
calibrators can influence the curve stability differently only
when a wrong weighting factor is used. When the correct
weighting factor of 1/x2 is used, the curve is always stable
regardless the calibrator spacing. (Please see Supporting
Information C for the detailed discussion.)
Data Quality. With the three established curves (using the
weighting factors of 1, 1/x, and 1/x2) for each individual run,
the QC concentrations were calculated, and QC data for run 3
are shown in Supporting Information D, Table SI 2a−2c. The
results showed that “good” STD curves generated “good” QC
data and “bad” STD curves generated “bad” QC data no matter
which weighting factor was used for the STD curves. As
discussed above, any STD curves generated using incorrect
weighting factors are not stable. Good accuracy could
sometimes also be achieved in cases when those curves were
“luckily” overlapped with or very close to the corresponding
STD curves generated with the correct weighting factors (Table
3d). Therefore, a STD curve with good accuracy or even both
the STD curve and QC data with good accuracy does not
necessarily mean that a correct weighting factor has been used,
and this should not be used as the criteria in selecting of
weighting factor. Fortunately, even with the incorrect weighting
factors, there was no or very insignificant impact on the
concentration data reported because the concentrations were
always reported with passing curves which actually overlapped
with or were very close to the curves using the correct
weighting factor, as shown in Table 3d. One thing worth
mentioning is that, in some cases, “bad” QC data could also be
generated with a “good” STD curve using a correct weighting
factor. This is almost always caused by some differences
between QC and STD samples, such as the differences between
storage conditions, preparation procedures, and matrix for STD
and QC samples, and therefore, QC performance should not be
used in the selection of weighting factor.
Assay Performance. However, the use of incorrect
weighting factors did impact the assay performance. A survey

Table 3a. STD Instrument Responses for BMS-708163 in Mouse Plasma LC-MS/MS Assay Validation

concn (ng/mL) 0.2 0.4 0.8 4 20 100 160 200

run 1 0.045576 0.090377 0.179375 0.886359 4.312755 20.806649 32.974830 41.307287
0.048468 0.097604 0.181369 0.905368 4.477241 21.436947 34.527598 42.261225
run 2 0.052042 0.096329 0.187270 0.864844 4.360585 21.658589 33.931792 41.885419
0.056547 0.104977 0.197225 0.920856 4.642914 23.277822 36.537529 46.105747
run 3 0.051634 0.095067 0.190177 0.891820 4.376717 22.056011 33.947702 43.279825
0.054632 0.098076 0.190476 0.926106 4.703780 22.764870 36.046021 46.925214

8963 dx.doi.org/10.1021/ac5018265 | Anal. Chem. 2014, 86, 8959−8966

Analytical Chemistry Article

Table 3b. Linear Regression Results for STD Curves in BMS-708163 in Mouse Plasma LC-MS/MS Assay Validation (Original
Data)
run 1 (Dev%a) run 2 (Dev%a) run 3 (Dev%a)
weighting factor weighting factor weighting factor
concn (ng/mL) 1 1/x 1/x2 1 1/x 1/x2 1 1/x 1/x2
0.2 −162.8 −14.3 −5.6 −125.5 −9.1 −6.2 2.2 −5.0 −3.1
0.2 −155.9 −7.4 1.1 −115.3 1.1 3.9 8.9 1.8 3.6
0.4 −78.0 −3.9 −0.9 −62.5 −4.5 −3.5 −0.2 −3.8 −3.1
0.4 −69.3 4.6 7.5 −52.7 5.3 6.2 3.1 −0.4 0.3
0.8 −35.9 0.9 1.1 −29.6 −0.8 −0.7 3.2 1.4 1.5
0.8 −34.7 2.0 2.3 −24.0 4.8 4.8 3.4 1.6 1.6
4 −2.8 4.1 2.1 −9.0 −3.6 −4.2 −0.7 −1.0 −1.5
4 −0.5 6.4 4.3 −2.7 2.8 2.1 3.1 2.8 2.4
20 1.2 2.2 −0.2 −2.5 −1.7 −2.5 −2.0 −2.1 −2.6
20 5.1 6.1 3.6 3.9 4.7 3.9 5.3 5.3 4.7
100 −1.0 −1.2 −3.6 −2.0 −2.1 −2.9 −1.1 −1.1 −1.7
100 2.0 1.8 −0.7 5.4 5.2 4.3 2.1 2.1 1.5
160 −1.8 −2.1 −4.5 −3.9 −4.1 −4.9 −4.9 −4.9 −5.4
160 2.8 2.5 0.0 3.5 3.2 2.4 1.0 1.0 0.5
200 −1.6 −1.9 −4.3 −5.1 −5.3 −6.1 −3.0 −3.0 −3.5
200 0.7 0.3 −2.1 4.5 4.2 3.4 5.2 5.2 4.6
a
Results expressed in percentage deviation from nominal concentrations.

Table 3c. Curve stability test using STD curves in BMS-708163 in mouse plasma LC-MS/MS assay validation
run 1 (Dev%a) run 2 (Dev%a) run 3 (Dev%a)
weighting factor weighting factor weighting factor
concn
(ng/mL) 1 1/x 1/x2 1 1/x 1/x2 1 1/x 1/x2
0.2 −720.9 −23.2 −6.3 −288.6 −18.9 −7.5 −43.5 −5.7 −3.1
0.2 −713.6 −16.0 0.5 −277.8 −8.1 2.9 −36.8 1.1 3.6
0.4 −353.8 −6.3 −0.4 −141.1 −6.6 −2.6 −22.8 −4.0 −3.1
0.4 −344.7 2.7 8.1 −130.7 3.7 7.4 −19.5 −0.6 0.3
0.8 −170.7 1.8 2.2 −65.8 0.9 1.2 −7.9 1.5 1.6
0.8 −169.4 3.0 3.4 −59.9 6.8 6.9 −7.7 1.7 1.7
4 −24.8 7.7 3.7 −11.7 0.9 −1.6 −2.5 −0.8 −1.3
4 −22.4 10.0 5.9 −5.0 7.6 4.9 1.3 3.1 2.5
20 1.6 6.2 1.4 1.7 3.5 0.4 −2.0 −1.7 −2.4
20 5.8 10.2 5.3 8.5 10.3 6.9 5.4 5.6 4.9
100 3.7 2.7 −2.0 −6.8 (19.5) −7.1 (19.5) −10.0 (19.5) −0.7 −0.8 −1.5
100 6.9 5.8 0.9 11.3 10.9 7.4 2.5 2.4 1.7
160 3.2 1.8 −2.9 −10.2 (30.0) −10.7 (30.0) −13.5 (30.0) −4.5 −4.5 −5.2
160 8.1 6.5 1.6 9.5 8.8 5.4 1.4 1.4 0.6
200 3.6 2.0 −2.7 −11.3 (37.0) −11.8 (37.0) −14.6 (37.0) −2.6 −2.6 −3.4
200 −13.6 (34.5) −14.8 (34.5) −18.8 (34.5) 10.6 9.9 6.4 4.0 (46.2) 3.9 (46.2) 3.2 (46.2)
curve extremely unstable stable extremely unstable stable extremely unstableb stable
stability unstable unstable unstable
a
Results expressed in percentage deviation from nominal concentrations. bAlthough the curve with 1/x weighting is unstable, the instability is almost
invisible with only one small instrument response bias (∼1.5%) from one STD sample. In this case, the small instrument response bias was used to
test the curve stability for the curve without weighting. The curve instability for curves with 1/x weighting can only be detected with one or a couple
of big instrument response biases, such as the examples shown in the table for run 1 and 2. Note: instrument response biases listed in parentheses,
please see Table 3a for the original instrument responses.

was conducted on more than 200 successful LC-MS/MS
sample analysis runs, and the correct weighting factor is 1/x2
for all of the runs. By using the acceptance criteria for regulated
GLP bioanalytical assays,6 about 80% and 5−10% of the runs
would have been failed runs if the wrong weighting factors of 1
and 1/x, respectively, were used. This will eventually result in
the significant cost increase for bioanalytical LC-MS/MS assay
validation, assay transfer, and sample analysis. The curve
stability and assay performance drop quickly while the
weighting factor used moving away from the correct weighting
8964
factor. This is probably the reason that, although most likely,
the correct weighting factor should be 1/x2, 1/x is still being
used for quite a few assays as it is hardly to be noticed with
“Test and Fit” (more than 90% of curves still pass), while the
weighting factor of 1 is rarely used as the 80% of curve failure
rate is easily noticeable.
Using Curve Stability Test in Selecting of Correct
Weighting Factor. The stability of a linear STD curve to any
random variations from only one or a couple of STD samples is
a quick and reliable indicator to check if a correct weighting
dx.doi.org/10.1021/ac5018265 | Anal. Chem. 2014, 86, 8959−8966
Analytical Chemistry Article

Table 3d. Quantative comparison of curve stability using STD curves in BMS-708163 in mouse plasma LC-MS/MS assay
validation
run 1 original passing curve with 1/x2 weighting (y = 0.215830x + 0.004819)
% difference for the predicted % difference for the predicted
concentration at STD low end concentration at STD high end
original passing curve, 1/x weighting (y = 0.210535x + 0.009489) −9.2%a 2.5%a
passing curve with one assumed instrument response biasc, 1/x2 weighting −0.8%a 1.7%a
(y = 0.212305x + 0.005788)
failed curve with one assumed instrument response biasc, 1/x weighting −22.0%a (−15.0%)b 6.6%a (4.0%)b
(y = 0.202458x + 0.014473)
original passing curve with 1/x2 weighting
run 3 (y = 0.224190x+0.008166)
% difference for the % difference for the
predicted concentration predicted concentration at
at STD low end STD high end
original passing curve with no weighting (y = 0.223013x+0.006071) 5.4%a 0.5%a
passing curve with one assumed instrument response biasc, 1/x2 weighting (y = 0.223861x+0.008257) −0.1%a 0.1%a
failed curve with one assumed instrument response biasc, no weighting, (y = 0.221928x + 0.026574) −41.8%a (−43.8%)b 1.0%a (0.5%)b
a
% differences for the predicted concentration comparing to the original passing curve with the weighting factor of 1/x2. b% differences for the
predicted concentration comparing to the original curve with the same weighting factor of 1 or 1/x. cPlease see Table 3a and 3c for the assumed
instrument response bias.

factor is used even with just one STD curve, where the
calculation of σ is impossible. Among all of the STD curves
generated from a set of STD data within a single run using
different weighting factors, such as 1, 1/x, 1/x2, 1/x3, and so
forth, the curve with the best stability can be easily identified
with a single instrument response bias (>20% bias away from
the original curve) at the STD high end for over weighting at
the high end, such as use of 1, 1/x for the curve with the correct
weighting factor of 1/x2, as shown in Table 3c, or at the STD
low end for over weighting at the low end, such as use of 1/x3
for the curve with the correct weighting factor of 1/x2 (data not
considering the x weighting is widely adopted for bioanalytical
LC-MS/MS assays and is built in most of the commercial
software, our recommendation is that 1/x2 should be used for
all bioanalytical LC-MS/MS assays. For other nonlinear curves
using least-squares regression algorithm, such as 4PL and 5PL
curves in LBA assays, 1/y2 should be used. One exception is
when quadratic curves or other nonlinear curves are severely
bended, and the linear relationship between σ and y does not
hold anymore, the correct weighting factor should be selected
using the proposed qualitative or quantitative approach. (Please
see Supporting Information E for more detailed discussion.)

shown). As there is one more degree of freedom in determining
quadratic curves, the curve stability test is less sensitive for
quadratic curves. Therefore, the test should be applied on the
linear curves only.
Weighting Factor Using 1/x2 or 1/y2 and Boundary of
the Application. As we have discussed, the weighting factor
was used to normalize the importance of the instrument
response errors in the estimation of the best-fit curve. The
instrument response errors are related to y directly, instead of x.
Therefore, an empirical function between 1/σ2 and y can be
established using either the plot or the linearity indicator
approach. In most cases, the effects of using the weighting
factor of 1/x2 or 1/y2 (1/x or 1/y) are the same for
bioanalytical LC-MS/MS assays because only linear or
quadratic curves with very mild curvature (close to linear) are
used. Therefore, the empirical functions between 1/σ2 and y
can be exactly (for linear curves) or approximately (for
quadratic curves with very mild curvature) translated to the
same empirical functions between 1/σ2 and x. However, for
quadratic curves with strong curvature and for 4-parameter
logistic (4-PL) and 5-PL curves commonly used in ligand
binding assays, the empirical function between 1/σ2 and y
cannot be translated to the same empirical function between
1/σ2 and x. As a result, finding an empirical function between σ
and x becomes very difficult for nonlinear STD curves while a
linear relationship always exists between σ and y and the
weighting factor of 1/y2 can be used directly. Therefore, the
weighting factor of 1/y2 is commonly used for 4PL and 5PL
curves in ligand binding assays. As the 1/x2 and 1/y2 weightings
are equivalent for bioanalytical LC-MS/MS assays, and also
8965
■
CONCLUSIONS
The procedure for the selection of the right weighting factor for
linear or quadratic calibration curves with least-squares
regression algorithm in bioanalytical LC-MS/MS assay was
presented and demonstrated. The weighting factor of 1, 1/x or
1/x2 should be selected if the standard deviation of the
instrument response (σ) is a constant, σ2 is proportional to x,
or σ is proportional to x, respectively. Further investigations
showed that 1/x2 should be used for all LC-MS/MS
bioanalytical assays as a linear relationship existed between σ
and x. The most stable curve could be obtained when the
correct weighting factor was used, whereas curves using
incorrect weighting factors were unstable. It was also found
in this work that there was no or very insignificant impact on
the concentrations reported with STD curves using incorrect
weighting factors as concentrations were only reported with
passing curves which actually overlapped with or were very
close to the curve using the correct weighting factor. However,
the run success rates dropped by about 80% and 5% to 10% for
assays using the incorrect weighting factors of 1 and 1/x,
respectively, when 1/x2 should be used. Finally, 1/x2 weighting
was recommended for all bioanalytical LC-MS/MS assays, and
1/y2 weighting should be used for other types of assays,
especially for assays using nonlinear calibration curves. All of
the findings in this work can be generalized and applied in
other quantitative analysis techniques using calibration curves
with weighted least-squares regression algorithm.
dx.doi.org/10.1021/ac5018265 | Anal. Chem. 2014, 86, 8959−8966
Analytical Chemistry

■
Article

ASSOCIATED CONTENT (27) Dawes, M. L.; Gu, H.; Wang, J.; Schuster, A. E.; Haulenbeek, J. J.
Chromatogr. B 2013, 934, 1−7.
*
S Supporting Information
(28) Gu, H. et al. Bristol-Myers Squibb, Princeton, New Jersey.
Additional information as noted in text. This material is Unpublished work, 2013.
available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION
Corresponding Author
*E-mail: huidong.gu@bms.com. Fax: 609-252-6171. Tel.: 609-
252-7636.
Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
The authors thank Ms. Carol Gleason and Dr. Qin Ji for their
helpful comments and discussions.

■ REFERENCES
(1) Almeida, A. M.; Castel-Branco, M. M.; Falcao, A. C. J.
Chromatogr. B 2002, 774, 215−222.
(2) Tellinghuisen, J. Analyst 2007, 132, 536−543.
(3) Nagaraja, N. V.; Paliwal, J. K.; Gupta, R. C. J. Pharm. Biomed.
Anal. 1999, 20, 433−438.
(4) Mulholland, M.; Hibbert, D. B. J. Chromatogr. A 1997, 762, 73−
82.
(5) Miller, J. N. Analyst 1991, 116, 3−14.
(6) Guidance for industrybioanalytical method validation. http://
www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf (accessed
Sept 1, 2013).
(7) Guidelines for validation of bioanalytical methods. http://www.
ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/
2011/08/WC500109686.pdf (accessed October 21, 2013).
(8) Kiser, M. M.; Dolan, J. W. LC·GC Eur. 2004, 17, 138−143.
(9) Steliopoulos, P.; Stickel, E. Anal. Chim. Acta 2007, 592, 181.
(10) Jain, R. B. Clin. Chim. Acta 2010, 411, 270−279.
(11) Burrows, J. Bioanalysis 2013, 5, 959−978.
(12) Tan, A.; Levesque, I. A.; Levesque, I. M.; Viel, F.; Boudreau, N.;
Levesque, A. J. Chromatogr. B 2011, 879, 1954−1960.
(13) Handbook of LC-MS Bioanalysis: Best Practices, Experimental
Protocols, and Regulations; Li, W., Zhang, J., Tse, F. L. S., Eds.; John
Wiley & Sons, Inc.: Hoboken, New Jersey, 2013; pp 403−415.
(14) General description of homoscedasticity. https://en.wikipedia.
org/wiki/Homoscedasticity (accessed August 8, 2013).
(15) General description of heteroscedasticity. https://en.wikipedia.
org/wiki/Heteroscedasticity (accessed August 8, 2013).
(16) Gu, H.; Deng, Y.; Wang, J.; Aubry, A.; Arnold, M. J. Chromatogr.
B 2010, 878, 2319−2326.
(17) Clifford, A. A. Multivariate Error Analysis; John Wiley & Sons,
Inc.: Hoboken, New Jersey, 1973.
(18) Aitken, A. C. Proc. R. Soc. Edinburgh 1935, 55, 42−48.
(19) General description of least-squares. http://en.wikipedia.org/
wiki/Least_squares (accessed January 21, 2004).
(20) Engineering handbookdescription of weighted least squares
regression. http://www.itl.nist.gov/div898/handbook/pmd/section1/
pmd143.htm (accessed August 20, 2013).
(21) Ryan, T. P. Modern Regression Methods; John Wiley & Sons, Inc.:
Hoboken, New Jersey, 1997.
(22) Johnson, E. L.; Reynolds, E. L.; Wright, D. S.; PAchla, L. A. J.
Chromatogr. Sci. 1998, 26, 372−379.
(23) Karnes, H. T.; Shiu, G.; Shah, V. P. Pharm. Res. 1991, 8, 421−
426.
(24) Box, G. E. P.; Hunter, W. G.; Hunter, J. S. Statistics for
Experimenters, 1978, John Wiley & Sons, Inc.: Hoboken, New Jersey.
(25) Finney, D. J.; Phillips, P. J. Appl. Stat. 1977, 26, 312−320.
(26) Aubry, A.; Gu, H.; Magnier, R.; Morgan, L.; Xu, X.;
Tirmenstein, M.; Wang, B.; Deng, Y.; Cai, J.; Couerbe, P.; Arnold,
M. Bioanalysis 2010, 2, 2001−2009.

8966 dx.doi.org/10.1021/ac5018265 | Anal. Chem. 2014, 86, 8959−8966