RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2005; 19: 2131–2136

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2040

Development of a low volume plasma sample

precipitation procedure for liquid chromatography/

tandem mass spectrometry assays used for drug discovery

applications

Xiaoying Xu*, Qiao Zhou and Walter A. KorfmacherDepartment of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA

Received 25 February 2005; Revised 2 June 2005; Accepted 2 June 2005

The demand for high sensitivity bioanalytical methods has dramatically increased in the drug dis-

covery stage; in addition, there has been a growing trend of reducing the sample volume that is

required for these assays. A sensitive high-performance liquid chromatography/tandem mass spec-

trometry (HPLC/MS/MS) procedure has been developed and tested to meet these needs. The assay

requires only a low plasma sample volume (10mL) and employs a protein precipitation procedure

using a 1:6 plasma/acetonitrile ratio. The supernatant is injected directly into the LC/MS/MS system

using the selected reaction monitoring (SRM) procedure for detection. A generic HPLC gradient

based on a methanol/water mobile phase with a flow rate set to 0.8mL/min was used. The test meth-

od showed very good linearity between 0.1–1000ng/mL (R2¼ 0.9737), precision (%RSD¼ 6–9),

accuracy (%RE¼�2) and reproducibility (%RSD¼ 11). A drug discovery IV/PO study was assayed

using both the new low volume method and our standard volume (50mL) method. The correlation

of the two sets of data from the two methods was excellent (R2¼ 0.9287). This new assay procedure

has been successfully used in our laboratory for over 100 different rat or mouse discovery PK stu-

dies. Copyright # 2005 John Wiley & Sons, Ltd.

Many pharmaceutical companies are investing large

amounts of money in their research and development pro-

grams. The main driving force is the need to discover new

drugs that can cure health problems and be marketable to

allow the companies to eventually recover their investments

and make a profit. Drug metabolism and pharmacokinetics

(DMPK) research departments play a critical role in the

lead optimization phase of drug discovery by helping to

improve the potential candidate compound before it is

selected for development.1,2 Various absorption, distribu-

tion, metabolism and excretion (ADME) and pharmacoki-

netic (PK) screens are now key steps in early drug

discovery.1–5 Developing fast and cost-effective analytical

methods for ADME and PK studies has become a crucial

element for major pharmaceutical companies. Using high-

performance liquid chromatography combined with tandem

mass spectrometry (HPLC/MS/MS) has proven to be the

bioanalytical technique of choice for most of these ADME

and PK screens that are now used in the lead optimization

phase of drug discovery.1,4,6,7

During the drug discovery phase, the DMPK studies are

often performed in small rodents (mouse and rat). With

multiple requests from different assays (e.g., biological

testing, PK study, metabolism, toxicity, etc.), the available

plasma sample volumes for the bioanalytical analysis are

often very small.8 In recent years, the typical amount of

plasma that bioanalytical scientists have used for their assays

has dropped from 200 to 50 mL.9–13 Recent publications have

suggested that there is a need to decrease the sample volume

yet further.14–16 On the other hand, as new chemical entities

show increased potency, lower dosage of a drug in the

animals requires methods that have even higher sensitivity.

In the last decade, the limit of quantitation (LOQ) required for

a discovery PK bioanalytical assay was in the 10 ng/mL or

higher range.6,8 More recently, a LOQ of 1–10 ng/mL (based

on 50–200mL of plasma sample) has been needed for typical

drug discovery PK applications.7,17 For some highly potent

compounds, an LOQ of 0.1 ng/mL may be required even in a

drug discovery PK setting.

Another important aspect of drug discovery is that

discovery bioanalytical methods must be developed in a

short timeframe, with good levels of precision and a high

certainty of success. Protein precipitation is the simplest

approach for removing the majority of the protein

matrix.6,7,18 Protein precipitation has the advantage that it is

a generic procedure that works for most compounds; it has

the disadvantage that it results in a sample that is not as clean

as those produced by other extraction techniques. Fortu-

nately, because of the high selectivity of MS/MS with the

selected reaction monitoring (SRM) setting, protein precipi-

tation is normally an acceptable sample preparation proce-

dure for a discovery PK assay.6,7 By monitoring not only the

Copyright # 2005 John Wiley & Sons, Ltd.

*Correspondence to: X. Xu, Schering-Plough Research Institute,2015 Galloping Hill Road, K-15-2-2945, Kenilworth, NJ 07033,USA.E-mail: xiaoying.xu@spcorp.com

specific precursor mass of the drug ion but also a character-

istic product ion, interference from other matrix components

within the sample can be eliminated in most cases, although

matrix effects can still be an issue.6,7,17 Once an analytical

method has been developed, it is desirable that the method

performance remains reasonably consistent over time. The

results generated based on the established method should be

relatively free from systematic error and the relative error

should be kept to acceptable limits.7

Currently, for most higher-throughput bioanalytical

assays used in a drug discovery setting, 96-well plates are

used for the sample-handling procedures.7 These 96-well

plates have the advantage that they are relatively easy to

work with whether one is performing manual or robotic

sample-handling procedures. While some efforts have been

made to switch to 384-well plates for bioanalytical applica-

tions, these plates can be difficult to work with when the

operation is not fully automated. Therefore, our goal was to

demonstrate that one could use 96-well plates to perform

discovery PK assays with a 10-mL plasma sample in a routine

manner.

In this report, a new HPLC/MS/MS bioanalytical proce-

dure is described that is based on using very low volumes of

plasma samples (10mL). A partial validation of the procedure

was performed to ensure that the new method was robust

and reproducible. As a validation exercise, a drug discovery

IV/PO PK study was assayed using both the previous

standard method (based on a 50-mL sample volume) and the

new low volume procedure (based on a 10-mL sample

volume) to confirm the reliability of the new method.

EXPERIMENTAL

ReagentsFor sample preparation, acetonitrile (Otima; Fisher Scientific,

Pittsburgh, PA, USA) was used. For the high-performance

liquid chromatography (HPLC) mobile phase, methanol

(Optima; Fisher Scientific), ammonium acetate and acetic

acid (glacial, 99.99þ%; Aldrich Chemical Co., Inc., St. Louis,

MO, USA) were used. Deionized water was purified using a

compact ultrapure water system (EASYpure UV; Fisher

Scientific).

InstrumentationThe HPLC system consisted of Shimadzu (Columbia, MD,

USA) LC-10ADvp pumps with a Leap (Carrboro, NC, USA)

HTS PAL autosampler. A Phenomenex (Torrance, CA, USA)

Synergi Max reversed-phase HPLC column (C18,

30� 2.0 mm i.d., 5 mm) was used as the analytical column.

The mobile phase consisted of A (MeOH/H2O 20:80, 0.01 M

ammonium acetate, pH 6.0) and B (0.01 M ammonium acetate

in MeOHþ 0.6 mL/L 10% acetic acid, pH 6.0). A 1.5-min gra-

dient from 5% B to 95% B was employed at a flow rate of

0.8 mL/min. Under these conditions, the test compound

and the internal standard (IS) eluted at 0.97 and 1.02 min,

respectively. The HPLC eluant passed through a divert valve

and was then introduced directly into the source of the mass

spectrometer.

The HPLC/MS/MS measurements were performed using

a ThermoFinnigan (San Jose, CA, USA) Quantum triple-

quadrupole mass spectrometer. The mass spectrometer was

operated in the positive electrospray ionization (ESI) mode

with a spray voltage of 4000 V, capillary temperature of

3508C, sheath gas 80 psi, and auxiliary gas 20 psi. The MS/MS

measurements were performed using the SRM mode. The

SRM transitions were selected by subjecting the protonated

molecules to collision-induced dissociation (CID) in the

collision cell where the argon gas was maintained at a

constant pressure of 1.3 mTorr. The product ions were

monitored by scanning the third quadrupole of the MS/MS

system.6,7,17

Calibration standards, IS and quality controlsamples (QCs)In the standard volume 96-well plate procedure, the stock

solution of the test compound was prepared as a 1 mg/mL

solution in methanol and diluted with methanol to make

working solutions at 0.01, 0.1, 1, 10 and 100 ng/mL. The IS

was prepared in acetonitrile at a final concentration of

0.01 ng/mL. The low, medium and high QCs (1, 50,

1000 ng/mL) were prepared by spiking known quantities of

the working solutions (10–50 mL) into control rat plasma to

make a final volume of 1 mL and stored in a freezer at

�208C. The calibrators were prepared on the day of each

run. Calibration standards (0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25,

50, 100, 250, 500 and 1000 ng/mL) were prepared by spiking

known quantities of the working solutions (10–50mL) into

control rat plasma to make a final volume of 1 mL.

In the low volume procedure, the stock solution of the test

compound was prepared as a 1 mg/mL solution in methanol

and diluted with methanol to make the standard working

solutions at 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10

and 20 ng/mL. The IS was prepared in acetonitrile with a final

concentration of 0.01 ng/mL. The low, medium and high QCs

(1, 50, 1000 ng/mL) were prepared by spiking 5mL of the

appropriate standard working solution into 95mL of control

rat plasma to make a final volume of 100 mL and stored in a

freezer at�208C. The calibrators were prepared on the day of

each run. Calibration standards (0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10,

25, 50, 100, 250, 500 and 1000 ng/mL) were prepared by

spiking 5mL of the appropriate working solution into 95mL of

control rat plasma to make a final volume of 100 mL.

Sample preparationSample preparation in the standard volume 96-well plate

procedure has been described previously.7,13 Briefly, aliquots

of the plasma samples (50mL) were placed into a Strata 96-

well plate (AHO-7193; Phenomenex, CA, USA) and 150 mL

of the IS solution (0.01 ng/mL of IS in acetonitrile) were added

to each well (the protein precipitation ratio was 1:3). After

vortexing for 30 s, the plate was centrifuged (Eppendorf

5810; Westbury, NY, USA) for 10 min at 4000 rpm. Finally,

the supernatant was transferred to a second standard 96-

well plate (AHO-7192; Phenomenex) using the Tomtec

(Hamden, CT, USA) Quadra 96 instrument. For the assay,

an aliquot of the supernatant (5 mL) was injected into the

HPLC/MS/MS system for the assay.

Sample preparation using the low sample volume proce-

dure required only 10 mL of the plasma samples. The plasma

samples (e.g., standards, QCs, study samples) were placed

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2131–2136

2132 X. Xu, Q. Zhou and W. A. Korfmacher

into a v-bottom 96-well plate (AB-1058; ABgene House, UK)

and then IS solution (60mL; 0.01 ng/mL of IS in acetonitrile)

was added to each well (the protein precipitation ratio was

1:6). After vortexing for 30 s, the plate was centrifuged

(Eppendorf 5810) for 10 min at 4000 rpm. Finally, the super-

natant was transferred to a second v-bottom 96-well plate

(AB-1058) using the Tomtec Quadra 96 instrument. An

aliquot of the supernatant (5 mL) was injected into the

HPLC/MS/MS system for the assay.

Method testingThe quantitation range was from 0.1–1000 ng/mL. Two cali-

bration curves over this range were prepared on three sepa-

rate days. Each analytical accuracy and precision run

included calibration standards in duplicate within the 0.1–

1000 ng/mL range. The low, medium and high QCs (1, 50

and 1000 ng/mL) were assayed in replicates of six on three

separate days.

RESULTS AND DISCUSSION

The limit of quantitation (LOQ)The standard sample preparation method routinely used in

the laboratory is protein precipitation with a sample-to-sol-

vent ratio of 1:3. The test compound, blank control plasma,

0.1 ng/mL and 0.5 ng/mL standard plasma samples were

prepared via the standard volume (50mL plasma) or the

new low volume (10 mL plasma) sample preparation proce-

dure. After injecting these samples into the HPLC/MS/MS

system, the resulting mass chromatograms are shown in

Figs. 1–3. The expected retention time for the test compound

was 0.97 min.

In control plasma, the standard volume (50mL plasma)

procedure (protein precipitation 1:3 ratio) gave a background

peak intensity of 4.00 E2 counts per second (cps) (Fig. 1).

Using the low volume (10mL plasma) sample preparation

procedure (protein precipitation 1:6 ratio) showed a ten-fold

lower background intensity of 3.18 E1 cps (Fig. 1), which

clearly indicated that the low volume procedure resulted in a

significantly reduced level of background interference for

this test compound.

Analysis of the prepared 0.1 and 0.5 ng/mL standard

plasma samples by HPLC/MS/MS following the two sample

preparation procedures resulted in an impressive difference

between the two procedures. As shown in Fig. 2(A), there was

significant background interference and the compound was

not baseline separated from the chemical noise at the 0.1 ng/

mL concentration. As shown in Fig. 3(A), at 0.5 ng/mL, the

signal-to-noise (S/N) ratio achieved was 10 and it was set as

the LOQ for the standard volume (50mL plasma) sample

preparation procedure. When the same samples (0.1 and

0.5 ng/mL) were analyzed by the new low volume (10mL

plasma) sample preparation procedure, the results were

improved compared to the standard procedure. As shown in

Fig. 2(B), at the 0.1 ng/mL concentration of the test

compound, a S/N ratio of 10 was obtained, which resulted

in a five-fold lower LOQ for this assay. Even though the

absolute peak intensity of the test compound was smaller in

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

eA

bund

ance

RT: 0.97

RT: 0.97

NL: 4.00E2 (1: 3)

NL: 3.18E1 (1: 6)

Figure 1. Normalized mass chromatograms of blank control

plasma obtained using protein precipitation ratios of 1:3 and

1:6.

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Time (min)

0

20

40

60

80

100

Rel

ativ

eA

bund

ance

RT: 0.99

NL: 4.85E2

RT: 0.97

NL: 3.84E2

0

20

40

60

80

100

A (1:3)

B (1:6)

Figure 2. Normalized mass chromatograms of test com-

pound (0.1 ng/mL) in plasma obtained using protein pre-

cipitation ratios of 1:3 (A) and 1:6 (B).

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Time (min)

0

20

40

60

80

1000

20

40

60

80

100R

elat

ive

Abu

ndan

ceRT: 0.98

NL: 1.62 E3

RT: 0.97

NL: 8.81 E2

A (1:3)

B (1:6)

Figure 3. Normalized mass chromatograms of test com-

pound (0.5 ng/mL) in plasma obtained using protein pre-

cipitation ratios of 1:3 (A) and 1:6 (B).

Low volume plasma sample precipitation procedure for LC/MS/MS 2133

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2131–2136

the new low volume sample preparation procedure than in

the standard volume sample preparation procedure, the new

procedure resulted in a much lower noise level, thereby

increasing the S/N ratio. For comparison, Fig. 3(B) shows the

results obtained with the new low volume (10mL plasma)

sample preparation procedure for the 0.5 ng/mL sample.

Therefore, even though the absolute analyte peak intensity

dropped by a factor of two (as would be expected due to the

higher dilution), the new low volume sample procedure

provided a much higher S/N ratio due to the decrease in the

matrix signal.

Linearity of the standard curvesThe low plasma volume calibration curve showed a very

good linear range up to 1000 ng/mL (Fig. 4). The curves

were fit by the linear regression method using a 1/x2 weight-

ing. The correlation coefficient (R2) was 0.9737, which met our

discovery analytical criteria.7

ReproducibilityThe reproducibility of the low volume assay was performed

using either stock solution or a plasma standard with a con-

tinuous series of 96 and 192 injections, respectively. Figures 5

and 6 show the distribution of the injections vs. peak area

ratio of test compound and IS. The standard deviations

were 7 and 11%, respectively, which meets the criteria for

reproducibility of a bioanalytical assay.

Precision and accuracyThe precision of the method was defined as the percent rela-

tive standard deviation (%RSD) calculated from replicate

measurements. The accuracy of the assay was defined as

the percent relative error (%RE) of the mean of the replicate

measurements from the theoretical values. Precision and

accuracy were determined by analyzing QCs prepared at

three concentrations (1, 50, 1000 ng/mL) with six replicates

on three separate days. Tables 1 and 2 summarize the intra-

day and inter-day precision and accuracy for the new low

volume assay. The intra-day precision and accuracy were

between 5 to 6% and �7 to 6% (RE), respectively. The inter-

day precision and accuracy were between 8 to 9% and �4 to

3% (RE), respectively. All intra- and inter-day precision and

accuracy values were acceptable and spanned the entire con-

centration range, which indicated the assay was very robust,

reproducible and could be used to accurately quantify the

biological samples in a drug discovery environment.

ApplicationThe new low volume method was tested and compared with

the previous standard volume method in one drug discovery

PK study. The plasma samples were collected at 0.117, 0.25,

0.5, 1, 2, 4, 6, 8, and 24 h post-IV dose and at 0.25, 0.5, 1, 2, 4,

6, 8, 24 h post-PO dose. There were three animals in the IV and

PO groups, respectively. A total of 51 plasma samples (27 IV

and 24 PO samples) were analyzed under both the standard

and the low volume methods. Figures 7 and 8 show represen-

tative plasma concentration time profiles after IV and PO dos-

ing in animals using the standard and the low volume

methods. In both IV and PO dosages, the same PK profiles

were observed regardless of the sample preparation proce-

dure. Figure 9 shows that a very good correlation of the

y = 0.00156x - 0.0006R2 = 0.9737

0.0

0.4

0.8

1.2

1.6

2.0

0 200 400 600 800 1000 1200

Concentration (ng/mL)

Pea

kA

rea

Rat

io

Figure 4. Calibration curves of test compound (0.1–

1000 ng/mL) using the low volume method.

0

1

2

3

4

5

0 10 20 30 40 50 60 70 80 90 100

Injection

Rat

io

Figure 5. Reproducibility of the stock solution of a test compound using the low volume method.

2134 X. Xu, Q. Zhou and W. A. Korfmacher

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2131–2136

Table 2. Inter-day precision and accuracy of test compound

in rat plasma in the low volume assay (n¼ 6 per day, 3 days)

Precision (%RSD) Accuracy (%RE)

1 ng/mL 9 �450 ng/mL 8 �31000 ng/mL 9 3Mean 9 �2

Table 1. Intra-day precision and accuracy of the test

compound in rat plasma in the low volume assay (n¼ 6)

Precision (%RSD) Accuracy (%RE)

1 ng/mL 5 �750 ng/mL 6 61000 ng/mL 6 �5Mean 6 �2

0

1

2

3

4

5

0 20 40 60 80 100 120 140 160 180 200

Injection

Rat

io

Figure 6. Reproducibility of plasma standard of a test compound using the low volume method.

1

10

100

1000

0 5 10 15 20 25 30Time (h)

Plasma concentration (ng/mL)

Standard method

Low volume method

Figure 7. Representative plasma concentration time profile

after IV dose of a test compound in animal #2 (standard

method results vs. low volume method results).

1

10

100

1000

0 5 10 15 20 25 30Time (h)

Plasma concentration (ng/mL)

Standard method

Low volume method

Figure 8. Representative plasma concentration time profile

after PO dose of a test compound in animal #4 (standard

method results vs. low volume method results).

y = 1.0762x - 5.2276R2 = 0.9287

0

100

200

300

400

500

0 100 200 300 400 500Regular Plate

v-B

ott

omP

late

Figure 9. Correlation of the plasma concentrations (n¼ 51)

after IV and PO doses using the standard method (regular

plate) vs. the low volume method (v-bottom plate).

Low volume plasma sample precipitation procedure for LC/MS/MS 2135

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2131–2136

plasma concentrations (n¼ 51) was found for the two

methods; the correlation coefficient (R2) was 0.9287.

This new assay procedure has been successfully used in

our laboratory for over 100 different rat or mouse discovery

PK studies. The main advantage of this new low volume

assay is that smaller sample volumes can be utilized. We have

found this to be very helpful for discovery PK studies based

on the mouse in which study sample volumes are often small.

A second advantage of this new low sample volume

procedure is that only 10% of the control plasma is required

to make the calibration standards. The previous procedure

required 14 mL of control plasma for the calibration

standards while this new procedure requires only 1.4 mL of

control plasma—this translates into a significant saving when

a laboratory assays hundreds of discovery PK studies each

year.

CONCLUSIONS

The demand for high sensitivity bioanalytical methods,

coupled with a current trend of reducing the sample volume,

has dramatically increased in the drug discovery stage. As

drugs become more potent, the level of sensitivity of a bioa-

nalytical method has to be continuously increased and a low-

er LOQ is then required. On the other hand, the available

sample volume has often decreased so that a method based

on 50 mL of plasma is no longer preferred. Therefore, a low

sample volume (10 mL) HPLC/MS/MS method was devel-

oped to meet these needs. The new method was tested and

partially validated to ensure that the method’s performance

remained reasonably consistent over time and had acceptable

reproducibility. The good reliability and robustness of the

procedure was demonstrated using a drug discovery PK

study. Not only did this method provide excellent sensitivity

and linearity for quantitation of biological samples, but also it

reduced the control plasma costs to conduct the study due to

the lower volume of control plasma required for the calibra-

tion curve preparation.

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Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2131–2136