enhanced detection in capillary electrophoresis: example determination of serum mycophenolic acid

Research Article

Enhanced detection in capillaryelectrophoresis: Example determination ofserum mycophenolic acid

In order to overcome the poor absorbency detection limits in CE, two simple strategies

were combined to increase the amount of the sample injected: a long capillary to hold

extra sample while leaving adequate room for the separation and acetonitrile stacking,

which concentrated the sample based on transient pseudo-ITP. The combination of these

two strategies yielded sensitivity comparable or better than that of the HPLC with good

separation and with better theoretical plate number. The analysis of mycophenolic acid, a

common immunosuppressant drug, in serum was used here as an example to illustrate

this enhanced detection and its applicability to therapeutic drug monitoring. Acetonitrile

was used to remove serum proteins followed by direct injection filling 5–21% of the

capillary volume and separation in a borate buffer. The overall CE method compared well

to an assay by HPLC as far as sample preparation, correlation coefficient, and especially

sensitivity of detection.

Keywords:

HPLC / Immunosuppressant / Mycophenolic acid / Stacking / Therapeutic druglevel DOI 10.1002/elps.200800587

1 Introduction

One of the main practical shortcomings of CE in routine

analysis is the poor absorbency detection limits, which are

about 20–60 times less sensitive compared with HPLC. One

of the simplest ways to improve this is by concentration on

the capillary-stacking. Many types of stacking methods have

been described; however, the majority of them are limited in

applicability to a few compounds [1]. Two types of stacking

are of general use. One is the high field strength [1]. This

type is simple and can be applied to most compounds;

however, in practice it yields modest, 3- to 5-fold,

concentration on the capillary. A second type of stacking is

the acetonitrile or transient pseudo-ITP [1, 2]. This type is

also a general stacking, applicable to many different

compounds, easy to accomplish; but it can yield 5- to

25-fold increase in sensitivity. It occurs in samples contain-

ing acetonitrile and salts [2].

Acetonitrile is often used in CE and HPLC as a simple

method for serum deproteinization. Interestingly, acetoni-

trile and other organic solvents, present in the sample, exert

an opposite effect in HPLC compared with that in CE [3]. In

HPLC they cause band broadening due to the formation of a

short gradient; while in CE they cause increased apparent

theoretical plate number because of the stacking effect.

Acetonitrile (in the sample) in CE serves the function of a

terminating ion by supplying the high field strength

necessary to drive or increase the velocity of the analytes

(thus acting as a pseudo-terminating ion in ITP). The

common chloride ion (or other small ions) present naturally

in the deproteinized sample migrates rapidly, acting as a

leading ion producing a low field strength region. The

analytes stack temporarily between the leading ion and the

acetonitrile, analogous to that in ITP, before they enter

the separation buffer [2]. Several workers have confirmed

this type of stacking for different compounds varying from

drugs to peptides [4–10]. However, this stacking lacks the

basic and theoretical studies necessary to maximize its full

potential. One aim of this work here is to draw attention to

the importance of this simple stacking for routine analysis

using mycophenolic analysis as an example. The stacking by

itself improves greatly the detection limits, but it falls short

compared with that of HPLC. However, it is demonstrated

here that the sensitivity of detection, using mycophenolic

acid (MPA) as an example, can be enhanced much further

by combining this stacking with a longer capillary to hold an

extra large volume of the sample, while leaving ample room

in the capillary for adequate separation.

MPA is an immunosuppressant drug derived from the

fungus Penicillium stoloniferuman. It prevents rejection in

Zak K. Shihabi

Department of Pathology, WakeForest University, Baptist MedicalCenter, Winston-Salem, NC, USA

Received September 10, 2008Revised November 18, 2008Accepted December 20, 2008

Abbreviation: MPA, mycophenolic acid

Correspondence: Dr. Zak K. Shihabi, Department of Pathology,Wake Forest University School of Medicine, Winston-Salem, NC27157, USAE-mail: [email protected]: +1- 336 -716-9944

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2009, 30, 1516–15211516

organ transplantation. It is given as mycophenolate mofetil

(Cell Cept) or as mycophenolate sodium (Myfortic). Both are

metabolized in the liver to the active moiety MPA. MPA

undergoes glucuronidation as the first step in its metabo-

lism. The glucuronide derivative is not pharmacologically

active. However, there are other minor metabolites that may

be active. MPA acts as an uncompetitive, selective and

reversible inhibitor of inosine monophosphate dehy-

drogenase type II expressed on activated lymphocytes, the

enzyme that controls the rate of synthesis of guanine

monophosphate in the de novo pathway of purine synthesis

in the proliferation of B and T lymphocytes. MPA is exten-

sively and tightly bound to human serum albumin, with the

average bound fraction in individuals with normal renal

function being 97.5%. Its clearance is highly dependent on

protein binding (restrictive clearance) with an apparent half-

life of about 18 h.

MPA is administered mostly in combination with other

immunosuppressive drugs such as cyclosporine , tacrolimus

and prednisolone. This drug is also used as a steroid sparing

treatment in immune-mediated disorders including immu-

noglobulin A nephropathy, lupus nephritis and small vessel

vasculitides. The side effects of MPA therapy include diar-

rhea, nausea, vomiting, infections, leukopenia and/or

anemia . Many physicians simply decrease a prescribed dose

of this drug until the gastrointestinal symptoms disappear.

Thus the routine monitoring of this drug remains under

debate [11]. On the other hand, because of the lack of a

relationship between the dose and its serum levels and a

lack of an association of MPA levels with adverse symptoms,

therapeutic monitoring of MPA is recommended by many

investigators. Under such conditions the risk for acute

rejection is minimized and the patient outcome after renal

transplantation is improved [12].

The therapeutic level in most studies when MPA is used

in combination with other immunosuppressive drugs is

1–3.5 mg/L (3.2–11.4 mmol/L) for liver and kidney trans-

plants for trough concentrations and 30–60 mg/h/L for area

under the curve. At levels above the therapeutic range, the

relative risk of developing infection or leucopenia increases

more than threefold [13, 14].

Several methods have been described to measure total

and free MPA concentration such as HPLC with ultraviolet

detection, as well as mass spectrometric detection [15–17].

An enzyme-multiplied immunoassay technique that can be

automated on the general chemistry analyzers has also been

described and is used in some centers for total MPA

concentration [18].

Because the therapeutic levels are relatively low, the CE

has not been well investigated for the assay of MPA. Unsalan

et al. [19] have used CZE after sample evaporation to improve

the sensitivity, while Carlucci et al. [20] have used MEKC for

MPA determination after protein removal with acetonitrile or

acids. To improve the detection level of this drug in serum,

stacking on the capillary is desirable as a simple method.

Unfortunately stacking in MEKC is difficult for routine

analysis [21]. Furthermore, the addition of organic solvents,

such as acetonitrile in the sample to remove the excess of

proteins, limits the amount of sample in MEKC to be injected

on the capillary to a few seconds or less than 0.5% of the

capillary volume [21, 22]. On the other hand, the presence of

acetonitrile in the sample in CZE allows much larger sample

to be injected leading to stacking. This also eliminates the

tedious steps of evaporation and reconstitution to concentrate

the drug in the sample. Here we describe a simple and

enhanced assay for MPA in serum based on simultaneous

stacking by acetonitrile and the use of a long capillary with

separation by CZE. It illustrates the stacking property and

simplicity of acetonitrile for analysis of small molecules by CE.

2 Materials and methods

2.1 Chemicals

MPA, hippuric acid and boric acid were obtained from

Sigma Chemicals (St. Louis, MO, USA).

2.2 Instrument

Quanta 4000 (Waters, Milford, MA, USA) equipped with

untreated capillary 50 cm � 75 mm (id) (or as specified).

The voltage was set 14.5 kV and the wavelength was at

214 nm. Samples were injected hydrodynamically (10 cm

height gravity) for 40 s (or as specified) and electrophoresed

for 12 min.

2.3 Electrophoresis buffer

Two grams of boric acid was dissolved in 100 mL of water,

pH 7.5. Aliquots containing 10 mL of methanol and 100 mg

of SDS were added to the buffer.

2.4 ZnSO4 (for HPLC analysis)

One volume of ZnSO4 (0.1 M) was mixed with one volume

of acetonitrile (stable at room temperature).

2.5 Standard

MPA, 3 mg/L of 1% sodium chloride.

2.6 Procedure of CE

An aliquot of 100 mL of serum (or standard)was vortex-mixed

with 200 mL of acetonitrile containing 2 mg/L of hippuric

acid (as an internal standard) in 500 mL micro-centrifuge

tube and centrifuged for 15 s at 13 000g. The supernatant

was injected on the capillary.

Electrophoresis 2009, 30, 1516–1521 CE and CEC 1517


2.7 HPLC

For comparison, MPA was analyzed by HPLC too on a CN

Zorbax column 150� 4.6 mm packed with 5 mm particles

(Agilent Technologies, Wilmington, DE, USA), which was

eluted with a solvent of 25% acetonitrile in 0.1% phosphoric

acid. Serum samples were vortex-mixed with two volumes of

ZnSO4 solution and centrifuged for 40 s at 13 000g and

20 mL of the supernatant was injected on the column, with

detection at 254 nm (Model 441, WatersAssociates). This

method is similar to that by Svensson et al. [17] and Bolon

et al. [15] except that a CN column in place of C18 was used

to reduce the need for acetonitrile in the elution solvent.

This method was linear between 0.5 and 8 mg/L (r 5 0.99)

with minimum detection (3� baseline noise) for standards

of 0.125 mg/L. The average recovery from serum at 2 mg/L

was 88 %, and the RSD for nine replicates at 2.0 mg/L was

3.4%.

3 Results and discussion

Figure 1 illustrates electropherograms of an MPA

standard prepared in 1% sodium chloride (2.7 mg/L), a

serum of a patient free from this drug and the same serum

spiked with an added standard of 2.7 mg/L. Spiking

with a value within the therapeutic range gives a peak

height with good sensitivity (Fig. 1). The absorbency at

214 nm was about 2.3 times higher than that at 254 nm.

The test was linear between 0.5 and 6.0 mg/L with

r 5 0.99. The RSD based on nine replicates was 1.9%. The

recovery of MPA 2.7 mg/L in serum was 93% relative to a

standard prepared in 1% NaCl (n 5 6). Because of the

stacking, standards should be prepared in 1% saline or in

serum free from this drug rather than in water. An increase

in the buffer pH increased the migration time of the

MPA by CZE without affecting appreciably the peak height.

The SDS was added, below its critical micellar concentra-

tion, to speed up the migration. Checking 12 patients

free from this drug as well as several pools of serum, there

were no appreciable interfering peaks with the same

migration of MPA. Some peaks or noise close to the MPA

peak, at less than 0.3 mg/L, can be detected in some

patients. Thus, values below 0.5 mg/L are reported as less

than 0.5 mg/L.

The analysis of MPA by the CE was compared

with that by HPLC. The correlation between the CZE and

the HPLC for 19 patients is good with r 5 0.96 (Fig. 2). The

analytical aspects of both methods are comparable

with those of sample handling and analysis time in this

study and in a previous one [20]. Figure 3 illustrates the

analysis of a standard and the same patient by both

CE and HPLC. Some patients have a peak close to the MPA

peak. Such patients demonstrated a similar peak in

HPLC (Fig. 3D). Whether this peak represents a

metabolite of MPA is not clear. It is known that MPA

undergoes metabolism into several metabolites. In order to

separate the compound close to the MPA in the CE

method, addition of 5% of methanol to the electrophoresis

buffer gives a slight separation, while the addition

of 10% methanol was necessary to give separation close to

baseline (Fig. 3). Low values were more difficult to analyze

by HPLC due to interferences in the serum and loss line-

arity by peak height in the isocratic separation. Unsalan et al.[19] have used CZE for MPA analysis but after

solvent concentration and evaporation. Here in this method

we avoided all these extra steps with much improved

sensitivity.

Figure 1. Electropherogram of (A) MPA standard 2.7 mg/L in 1%NaCl, (B) patient sample free from MPA, (C) the same serumafter addition of 2.7 mg/L MPA (M 5 mycophenolic acid;H 5 hippuric acid as an internal standard); 60 s injection.

Electrophoresis 2009, 30, 1516–15211518 Z. K. Shihabi


Contrary to the MEKC, acetonitrile in the sample

improves the peak height and also improves the separation

in the CZE through stacking [1,2]. Figure 4 shows the peak

height of a standard with 4, 40 and 80 s injection (filling 0.5,

4.8 and 9.7% of the capillary volume, respectively).

Compared with that with 4 s injection (close to that used in

the MEKC), about 10- and 20-folds increase in sensitivity,

respectively, can be observed due to the stacking (Fig. 4).

The capillary at 80 s is filled with 9.7% of its volume with

sample. Injecting large volumes of sample decreases the

effective length of the capillary resulting in less-efficient

separation for serum.

In order to inject a larger amount of sample on the

capillary, with adequate separation, a longer capillary

75 mm� 80 cm (1.7� the effective length of the previous

one) was tried (Fig. 5). Here, a serum sample spiked with

just 0.3 g/L of the MPA standard (Fig. 5B) can be

separated and detected, after filling the capillary to 8% of its

y = 0.9557xR20.9699 = Sy.x=0.31

0

1

2

3

4

5

6

7

0 1 2 3 4 5 6

CE mg/L

HP

LC

mg

/L

Figure 2. Correlation of the CZE with the HPLC for MPA.

Figure 3. Comparisons of the CZE and HPLC for a standard andpatient on MPA: (A) CE of the standard 2.5 mg/L, (B) a patient,1.7 mg/L MPA, (C) standard by HPLC and (D) the same patient inB by the HPLC (M 5 mycophenolic acid; H 5 hippuric acid as aninternal standard). Figure 4. Stacking of 2 mg/L standard of MPA: (A) 4 s injection

(0.5% of the capillary volume), (B) 40 s injection (4.8 % of thecapillary) and (C) 80 s injection (9.7% of the capillary) (M 5 myco-phenolic acid; H 5 hippuric acid as an internal standard).



volume, much better than on the previous capillary. In pure

standards, this capillary can be filled further to 21% of its

volume with sample. Thus, values below the therapeutic

range can be detected (Fig. 6). The limits of detection

depend on many factors such as the electronics, sample

loading, solvents, etc.; however, here the CE can attain the

same, if not better, sensitivity, compared with that of HPLC

(Fig. 6) (minimum detection level is 95 mg/L for CE versus125 mg/L for HPLC). In this case, the length of the injected

sample segment on the capillary was initially about 200 mm.

Remarkably, at the end of the electrophoretic step it

concentrated down to a few millimeters at the detector. The

concentration factor on this long capillary at 21% of the

capillary volume compared with the shorter one with 0.5%

injection represents about 70-fold. Also, in this case, the

apparent theoretical plate number for MPA peak is much

better by the CE compared with that by the HPLC, 53 265

versus 4620, respectively (Fig. 6).

4 Concluding remarks

Neither a long capillary by itself nor stacking by itself is

sufficient to bring along the detection limits of the CE to

that of HPLC. However, the combination of these two

simple strategies did, through an increase in the injected

sample volume. Traditionally, longer capillaries are used

mainly to improve the resolution in CE. However, they also

give increased theoretical plate number in addition to

allowing loading more sample volume. Here, a longer

capillary contributed better to the stacking process by

holding larger sample volume, while leaving enough room

for separation of the complex serum samples. An increase

Figure 5. MPA separation from serum on a long capillary(75 mm� 80 cm) with injection filling 8% of the capillary volume,(16 kV): (A) serum sample free from MPA (M), (B) the samesample but spiked with 300 mg/L of MPA and (C) standard of 700mmg/L MPA (M).

Figure 6. Comparison of the CE (top) and the HPLC (bottom) fora standard of 400 mg/L MPA (M) on a long capillary(75 mm� 80 cm). The standard in the CE was injected for 500 sfilling 21% of the capillary volume (16 kV).

Electrophoresis 2009, 30, 1516–15211520 Z. K. Shihabi


in the injected sample volume alone, under non-stacking

conditions, causes a rapid deterioration of the theoretical

plate number and the resolution too [23]. Acetonitrile in the

sample overcomes that, while inducing �20-fold concentra-

tion based on the described mechanism of transient pseudo-

ITP [2]. The wide capillary (75 mm id) chosen here,

compared with the more common 50 mm, also doubled the

signal. The combination of these two (or three) strategies

here offered suddenly a greater sensitivity, which matches

that of the HPLC (Fig. 6) (the minimum detection level is

95 mg/L for CE versus 125 mg/L for HPLC), also with better

theoretical plate number. The combination of acetonitrile

stacking with other techniques such as bubble cells

or Z cells probably will improve further the detection limits

too.

Acetonitrile stacking [13, 14], unfortunately,

has not been investigated very much. Many aspects of this

technique remain unknown and deserve further studies. In

order to fully optimize this technique, uncoupling

the stacking step from the separation step is needed.

Imaging techniques in this respect will be helpful. The

mathematical modeling of this stacking has not been

approached either.

After this paper has been submitted, another study [24]

showed that MPA and its glucauronide can be determined

by CE based on the acetonitrile stacking described here.

However, the authors utilized a short capillary with small

diameter (25–35 cm� 50 mm). As a result of this, the

stacking was modest but adequate.

Injecting large sample volumes also has its own short-

comings in terms of increasing the analysis time. The

present CE instrumentations are not designed to qinject

large volumes of samples rapidly in few seconds. The

injection time here, in some cases, increased too long to

500 s, which is not suitable in routine analysis. The capillary

wash and filling steps also require longer time. In most

instances, as for the MPA analysis, acetonitrile stacking in a

short capillary offers enough sensitivity for the needed

therapeutic range and offers the desired high speed. On the

other hand, some analytes are present in very low concen-

trations so that a longer capillary becomes necessary.

Hopefully, the next generation of CE instruments can be

designed to take advantage of the stacking methods needs.

In summary, the combination of a long capillary

and acetonitrile injection directly yielded an enhanced

sensitivity, which matches that of the HPLC, and with

better theoretical plate numbers; while eliminating

the use of expensive columns and avoiding the consump-

tion of high concentrations of organic solvents needed in

HPLC.

The author has declared no conflict of interest.

5 References

[1] Shihabi,Z., J. Chromatogr. A 2000, 902,107–117.

[2] Shihabi, Z., Electrophoresis 2002, 23, 1612–1617.

[3] Shihabi, Z., J. Liq. Chromatogr. Relat. Technol. 2008, 31,3159–3168.

[4] Choy, T. M., Chan, W. H., Lee, A. W., Huie, C. W., Elec-trophoresis 2003, 24, 3116–3123.

[5] Kong, Y., J. Liq. Chromatogr. Relat. Technol. 2006, 29,1561–1573.

[6] Kong, Y., Zheng, N., Zhang, Z., Gao, R., J. Chromatogr.B 2003, 795, 9–15.

[7] Sarah, Y. C., Nan-Yan, Z., Chee-Shan, C., Int. J. Appl.Sci. Eng. 2004, 2, 277–285.

[8] Kubalczyk, P., Bald, E., Anal. Bioanal. Chem. 2006, 384,1181–1185.

[9] Galeano-Diaz, T., Acedo-Valenzuela, M. I., Mora-Diez, N.,Silva- Rodriguez, A., Electrophoresis 2005, 26, 3518–3527.

[10] Shihabi, Z., J. Chromatogr. A. 1996, 744, 231–240.

[11] West-Thielke, P., Kaplan, B., Am. J. Transplant. 2007, 7,2441–2442.

[12] Meur, Y. L., Buchler, M., Thierry, A., Caillard, S., Ville-main, F., Lavaud, S., Etienne, I. et al., Am. J. Transplant.2007, 7, 2496–2503.

[13] Tredger, J. M., Brown, N. W., Adams, J., Gonde, C. E.,Dhawan, A., Rela, M., Heaton, N., Liver Transplantation2004, 10, 492–502.

[14] Van Gelder, T., Le Meur, Y., Shaw, L. M., Oellerich, M.,DeNofrio, D., Holt, C., Kaplan, B. et al., Ther. Drug Monit.2006, 28, 145–154.

[15] Bolon, M., Jeanpierre, L., El Barkil, M., Chelbi, K.,Sauviat, M., Boulieu, R., J. Pharm. Biomed. Analysis2004, 36, 649–651.

[16] Annesley, T. M., Clayton, L. T., Clin. Chem. 2005, 51,872–877.

[17] Svensson, J. O., Brattstrom, C., Sawe, J., Ther. DrugMonit. 1999, 21, 322–324.

[18] Shipkova, M., Schutz, E., Armstrong, V. W., Niedmann,P. D., Wieland, E., Oellerich, M., Transplant. Proc. 1999,31, 1135–1137.

[19] Unsalan, S., Hempel, G., Fobker, M., Wurthwein, G.,Boos, J., Chromatographia 2006, 64, 419–421.

[20] Carlucci, F., Anzini, M., Rovini, M., Cattaneo, D., Merlini,S., Tabucchi, A., Electrophoresis 2007, 28, 3908–3914.

[21] Shihabi, Z., in: Cazes, J. (Ed.), Encyclopedia ofChromatography, Taylor & Francis, Boca Raton 2005,pp. 1490–1498.

[22] Shihabi, Z., in: Landers, J. P. (Ed.), Handbook of Elec-trophoresis, 2nd ed. CRC Press, Boca Raton, FL 1997,pp. 457–477.

[23] Vinther, A., Soeberg, H., J. Chromatogr. 1991, 559, 3–26.

[24] Ohyama, K., Kinoshita, N., Kishikawa, N., Kuroda, N.,Electrophoresis 2008, 29, 3658–3664.



enhanced detection in capillary electrophoresis: example determination of serum mycophenolic acid

Documents