comparative evaluation of ce and hplc for determination of cotinine in human urine

Comparative Evaluation of CE and HPLCfor Determination of Cotinine in Human Urine

Piotr Kowalski&, Marcin Marszałł, Ilona Oledzka, Wojciech Czarnowski

Faculty of Pharmacy, Medical University of Gdansk, Hallera 107, 80-416, Gdansk, Poland; E-Mail: [email protected]

Received: 23 March 2007 / Revised: 28 May 2007 / Accepted: 15 June 2007Online publication: 10 August 2007

Abstract

Two rapid and popular methods—capillary electrophoresis (CE) and high-performance liquidchromatography (HPLC) have been compared for analysis of cotinine in human urine. Cotininewas analyzed in less than 7 min, with detection limits of 5 and 3.2 ng mL)1 for CE and HPLC,respectively. The performance of the methods was evaluated in terms of sensitivity, specificity,precision, accuracy, and limits of detection and quantification. Calibration plots were linear inthe range 50–4,000 ng mL)1, at least, and mean recoveries were satisfactory for bothtechniques. The methods were successfully used for quantification of cotinine in urine.

Keywords

Capillary electrophoresisColumn liquid chromatographyHuman urineCotinineValidation study

Introduction

Voluntary and involuntary cigarette

smoking is the main cause of a lung

cancer and a major factor in cardiovas-

cular diseases and chronic lung inflam-

matory disorders [1–5]. Tobacco smoking

is a habit which can be overcome. Many

clinical and epidemiological studies have

shown that prenatal exposure to tobacco

smoke has a significant affect on lung

function, asthma risk, and respiratory

infection [3, 6–8]. Most studies prove

exposure to tobacco smoke is an impor-

tant and preventable cause of morbidity

among children and pregnant women

[9–12]. Chemical smoke compounds,

including nicotine, carbon monoxide, and

tobacco-specific carcinogens can be de-

tected both in smokers and in non-

smokers who are exposed to cigarette

smoke [13–17]. Because smoking is a

worldwide problem, it is very important

to find simple and cheap methods for

estimating exposure to smoke.

In humans approximately 86% of the

nicotine absorbed from tobacco smoke is

metabolized to cotinine by C-oxidation

by hepatic enzyme cytochrome P450

(CYP) 2A6 [18, 19]. The half-life of nic-

otine is relatively short (2 h) whereas that

of cotinine is long (approx. 16–20 h)

[19–21]. As a consequence of the short

half-life of nicotine, the ratio of cotinine

to nicotine is highly dependent on the

time since last exposure to nicotine.

Quantitative analysis of cotinine in

physiological fluids (for example urine,

saliva, serum, and plasma) and hair can

be achieved by gas chromatography with

nitrogen–phosphorus or electron-capture

detection (GC–NPD, GC–ECD) [22], gas

chromatography with mass spectrometric

detection (GC–MS) [23–26], high-perfor-

mance thin-layer chromatography

(HPTLC) with densitometry [27], high-

performance liquid chromatography

(HPLC) with ultraviolet detection [9, 11,

27–31], enzyme-linked immunosorbent

assay (ELISA) [32], radioimmunoassay

(RIA) methods [33], and non-aqueous

capillary electrophoresis with electro-

chemical detection [34]. The combination

of solid-phase extraction with CE or

HPLC and MS detection for identifica-

tion of nicotine and its metabolites in

urine has also been described [35, 36].

Although MS is much more sensitive and

more specific than other methods of

detection, it has been applied less fre-

quently because of the high cost of the

instrumentation.

The main objective of the work dis-

cussed in this paper was to develop rapid,

simple, and low-cost methods which do

not involve complicated clean-up proce-

dures for determination of cotinine as a

biomarker in human urine. These proce-

dures have also been compared in routine

analysis of urine from smokers and non-

smokers. Although the all work cited

above enables determination of cotinine

2007, 66, 357–361

DOI: 10.1365/s10337-007-0331-60009-5893/07/09 � 2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

Original Chromatographia 2007, 66, September (No. 5/6) 357

at low levels, no fully validated electro-

phoretic method was available for quan-

tification of cotinine in urine.

Experimental

Reagents and Standards

All reagents were of analytical grade and

solvents were of chromatography purity.

Dichloromethane was obtained from

BDH Laboratory Supplies (Dorset, UK)

and acetonitrile from LabScan (Dublin,

Ireland). Highly pure water was obtained

from Milli-Q equipment (Millipore,

Bedford, MA, USA). Cotinine was pur-

chased from Sigma-Aldrich (St Louis,

MO, USA). The phosphate buffer solu-

tion for HPLC analysis was prepared

from dibasic sodium phosphate hepta-

hydrate (Sigma-Aldrich) and 85%

orthophosphoric acid (Riedel-de Haen,

Seelze, Germany). The buffer solution for

CE analysis was prepared from sodium

dihydrogen phosphate, boric acid

(Merck, Darmstadt, Germany), and 85%

orthophosphoric acid.

Stock solutions of cotinine

(25.0 mg mL)1) in methanol were

stored at )20 �C in sealed volumetric

flasks. Working solutions used for

studies of separation performance were

prepared in methanol. Control

solutions (50–4,000 ng mL)1 for both

methods) for generation of calibration

plots were freshly prepared in cotinine-

free human urine immediately before

analysis. Calibration plots were gener-

ated from at least seven points, each

point being the average from three

runs.

Instrumental Conditions

CE

Experiments were performed with a

Beckman P/ACE 2100 instrument

equipped with an autosampler, selectable

fixed-wavelength UV detector, and Gold

software for system control and data

collection. The capillary cartridge con-

tained a 75 lm i.d. unmodified silica

capillary, 57 cm total length and 51 cm

effective length to the detector. The po-

tential was maintained at 20 kV and the

capillary was thermostatted by means of

cooling fluid at 25 �C. The buffer solutionwas prepared by mixing 10 mM sodium

dihydrogen phosphate, 2 mM boric acid,

and concentrated orthophosphoric acid

in appropriate proportions to obtain a

final pH of 2.7. The capillary was regen-

erated between each run by treatment

with 0.1 M hydrochloric acid, then with

regeneration solution (0.1 M sodium

hydroxide), and, finally, with triple-dis-

tilled water.

HPLC

HPLC was performed with a P580 pump,

STH 585 column oven, and 340S UV

diode-array detector (all from Dionex,

USA). Evaluation and quantification

were performed with a Chromeleon

(version 6.20) chromatography manage-

ment system. Compounds were separated

on a 250 mm · 4.6 mm i.d., 5 lm parti-

cle, BDS C18 reversed-phase column

(Thermo Hypersil, UK). The mobile

phase was 0.03 M dibasic sodium phos-

phate heptahydrate containing 5% (v/v)

acetonitrile and adjusted to pH 3.2 with

85% orthophosphoric acid. Isocratic

elution was performed at 40 �C at a flow

rate of 1 mL min)1.

Sample Preparation

All urine samples were collected in non-

sterile polyethylene urine containers,

frozen, and stored at )20 �C until anal-

ysis. Non-smoker’s urine was spiked with

known amounts of cotinine standard to

prepare quality-control (QC) samples.

Urine pH and density were measured at

each sampling time with Multistix 10 SG

analytical tests (Bayer, Germany). Coti-

nine levels were standardized by correc-

tion for creatinine excretion, with results

expressed as cotinine-to-creatinine ratio.

Fig. 1. Typical chromatograms obtained from a blank urine, and b urine spiked with 2,340 ngmL)1 cotinine (1, tR 4.9 min)

358 Chromatographia 2007, 66, September (No. 5/6) Original

Before extraction, samples were left to

thaw and to equilibrate to room

temperature. Urine (2.5 mL) was placed

in an 8 mL polyethylene tube. Sodium

hydroxide (5 M, 0.5 mL) was added, then

2.5 mL dichloromethane, and the mix-

ture was shaken (rotary mixer) for

15 min. After centrifugation for 15 min

at 2,500 rpm the aqueous layer was dis-

carded and 1.5 mL organic phase was

transferred to an Eppendorf-type snap-

closure tube containing a solution of

hydrochloric acid in methanol (0.1%,

0.1 mL). The extract was evaporated in

stream of air and the residue was recon-

stituted in 0.1 mL HPLC mobile phase or

0.1 mL CE running buffer. Injection

volume for HPLC was 20 lL. For CE

analysis the samples were introduced

from the anodic end of the capillary by

vacuum injection for 2 s at 0.5 psi. All

urine samples were prepared in triplicate

for both methods.

To evaluate precision the method was

tested with real samples from 40 patients.

Subjects were classified in four groups

(smokers, substitute nicotine therapy,

passive smokers, and non-smokers) in

accordance with answers given in a

questionnaire. Pearson’s coefficient was

used to calculate the correlation between

amount of cigarette smoke and urinary

cotinine levels measured by CE and

HPLC. One-way analysis of variance

(ANOVA) was used to compare urinary

cotinine levels among active, passive, and

non-smokers for each of the three types

of measurement.

Validation Study

Appropriate validation is necessary to

ensure the suitability for purpose of

analytical methods. Both methods pro-

posed for determination of cotinine were

validated for specificity, linearity, limits

of detection and quantitation, precision,

and accuracy. Quality-control assessment

of the methods for determination of

urinary cotinine concentrations was per-

formed at three concentrations 50, 500,

and 2,000 ng mL)1 (QC solutions).

Within-run (intra-assay) precision of

both methods was evaluated by analysis

of the same urine samples ten times,

independently prepared, in the same

sample set. Between-run (inter-assay)

precision was determined by analysis of

the same urine samples on ten consecu-

tive days. Inter and intra-day variation

were assessed using the QC solutions. The

limits of detection (LOD) and quantifi-

cation (LOQ) were estimated as the

amounts of analyte for which the peak

height or peak area was three and ten

times the signal-to-noise ratio, respec-

tively (i.e. S/N = 3 or 10). Mean recov-

ery from urine of the analyte at the three

QC levels was calculated by comparing

the concentrations measured in the coti-

nine-supplemented urine with the con-

centration actually added. Statistical

analysis, i.e., determination of linear

regression data, intercept, and slope, was

performed by use of Statistica for

Windows (Statistica 7.1; Statsoft, 2006).

Results and Discussion

This paper highlights the possibility of

application of both CE and HPLC to

identification and quantification of coti-

nine in human urine. The sensitivity,

linear range, detection and quantitation

limits, reproducibility, accuracy, and

Fig. 2. Typical electropherograms obtained from a blank urine, and b urine spiked with 2,500 ngmL)1 cotinine (1, tM 5.0 min)

Table 1. Summary of precision and validation data for analysis of cotinine by HPLC and CE

HPLC CE

Linear range (ng mL)1) 50–4,000 50–4,000Slope ± SD )0.0788 ± 0.005 0.2663 ± 0.003Intercept ± SD 40.09 ± 0.58 14.96 ± 7.39Correlation coefficient 0.9997 0.9995N 7 7LOD (ng mL)1) 3.2 5LOQ (ng mL)1) 10 15Total separation time (min) 7 7Cotinine retention or migration time 4.8 5.2


precision of the methods were satisfac-

tory. The specificity of both methods was

confirmed by analysis of different blanks

and extracts (n = 6) from urine. Chro-

matograms obtained from extracts of

blank urine and urine spiked with coti-

nine are shown in Figs. 1a, b, respec-

tively. Electropherograms obtained from

the same samples are shown in Figs. 2a,

b, respectively. Figure 1a and 2a show

there was no interference in the region

where the analytes eluted.

Regression data for calibration plots,

and limits of detection and quantifica-

tion, are listed in Table 1. The precision

of the HPLC assay, for intra-day vari-

ability, ranged from 4.8% for 2,000 ng

mL)1 to 7.1% for 500 ng mL)1, while in

the case of CE, ranged from 2.7% for

2,000 ng mL)1 to 9.6% for 50 ng mL)1.

The intermediate precision expresses

within-laboratory variation of results

from analysis by different analysts on

different days with newly prepared sam-

ples, buffer solution for CE, and mobile

phase for HPLC. Independent assays

performed by two analysts on different

days showed the repeatability and

reproducibility of the methods was good

(Tables 2, 3). Good recovery was ob-

tained for the analyte for all spike levels,

and average recoveries complied with the

requirement they should be >90%.

Recovery for the CE method varied from

91.8 to 98.3% (RSD 3.5%) whereas that

for the HPLC method was from 89.4 to

98.5% (RSD 4.7%) (Table 4).

Analysis of urine enables identifica-

tion of biomarkers for a variety diseases

of the kidney or urogenital tract [37]. One

problem with urine analysis is that its

polypeptide composition changes sub-

stantially during the day, most probably

as a consequence of physical activity,

diet, or smoking. Urine contains inor-

ganic ions and other endogenous com-

pounds, for example urea, that can also

interfere with analysis [38]. The high salt

content of crude urine samples results in

increased analysis times and also sub-

stantial peak broadening in HPLC and

CE analysis. It is, therefore, essential to

remove both salts and other low-molec-

ular weight compounds to avoid capillary

breakage because of currents which are

too high. Among sample-preparation

techniques, liquid–liquid extraction

(LLE) is an efficient clean-up procedure

for removing unwanted substances from

the urine matrix; it can also be used to

concentrate the analyte. In addition to

the sample clean-up procedure proposed,

alkaline hydrolysis of analyte conjugates

is also necessary. The glucuronide con-

jugates were determined indirectly by

initial basic hydrolysis of urine sample

followed by quantification. Cotinine has

several advantages over other biochemi-

cal markers as an objective indicator of

nicotine intake [39]. Urinary cotinine

levels were higher for all measurements

among active smokers and lowest among

non-smokers. Urinary cotinine levels

measured by both methods are compared

in Table 5. Cotinine levels were corre-

lated with the number of cigarettes

smoked per day or with substitute nico-

tine therapy. Pearson’s coefficient (r) was

0.903 and 0.907 for CE and HPLC,

respectively.

Comparative Study

In general terms, both methods use a

simple procedure for extraction of coti-

nine and are based on readily available

chemicals and conventional instrumenta-

tion. The short analysis time (7 min) and

appropriate resolution between cotinine

and impurities were achieved by selection

of optimum electrophoretic and chro-

matographic conditions. Consumption of

organic solvent in the CE method was

much lower, which is of great economic

benefit. By use of appropriate validation

tests we proved both methods gave simi-

lar results. Between-day variability is

better for CE whereas sensitivity and

within-day variability are better for

HPLC. Comparison of the two tech-

niques has also shown the first is more

Table 2. Assay-validation results obtained from within-run experiments on analysis of cotinine by HPLC and CE

Nominalconcentration(ng mL)1)

Within-run (repeatability, 1 day)

Measuredconcentrationa

(ng mL)1)

Precision, asRSD (%)

Accuracy, asrecovery (%)


(ng mL)1)


Accuracy,as recovery (%)

HPLC method CE method

50 46.2 7.7 92.3 45.9 ± 4.4 9.6 91.8500 472.5 7.1 94.5 470.7 ± 23.5 5.0 94.1

2,000 1982 4.8 99.1 1965.1 ± 53.9 2.7 98.3

a Mean concentration ± SD in human urine samples (n = 6)

Table 3. Assay validation results obtained from between-run experiments on analysis of cotinine by HPLC and CE

Nominalconcentration(ng mL)1)

Between-run (intermediate precision)

Measuredconcentrationa (ng mL)1)




(ng mL)1)




50 43.3 7.8 86.5 47.1 ± 5.7 12.1 94.3500 465.5 5.2 93.1 465.7 ± 24.4 5.2 93.1

2,000 1966 4.8 98.3 1961.3 ± 56.5 2.9 98.1

a Mean concentration ± SD in human urine samples (n = 6)

360 Chromatographia 2007, 66, September (No. 5/6) Original

accurate and reproducible whereas the

latter is more sensitive and selective.

Precision at very low levels was better for

HPLC but separation efficiency is better

for CE, which also has practical and

economic advantages. Although the pro-

posed HPLC method was more sensitive,

CE seems be more convenient for routine

analysis, because in the long term it is

more cost-effective than HPLC.

Conclusions

Comparison of the CE and HPLC meth-

ods revealed the latter requires muchmore

organic solvent whereas CE is more ver-

satile and less expensive. The HPLC

method is more sensitive, precise, and en-

ables better resolution of cotinine in urine

samples. Both assays are rapid and require

relatively simple sample preparation. It

was apparent from the results there were

no significant differences between the

techniques for cotinine determination.

These methods have great potential be-

cause urine is a biological fluid readily

obtainable by use of non-invasive collec-

tion procedures. Likewise, the results ob-

tained in the validation process and in

cotinine analysis are encouraging for both

techniques, although there are a slight

differences between the results obtained,

and indicate their suitability for routine

analysis in any clinical laboratory. Despite

the huge efforts of many researchers to set

up different (and often expensive) new

techniques, HPLC and CE with UV-

detection remain simple ways of achieving

inexpensive and rapid analysis.

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Table 4. Results from recovery study

Nominal concentration(ng mL)1)

Recovery for both methods

Mean recovery(%) (n = 6)

Overallrecovery (%)

OverallRSD (%)

Meanrecovery (%)(n = 6)

Overallrecovery (%)

OverallRSD (%)


50 89.4 94.4 4.7 91.8 94.7 3.5500 95.2 94.1

2,000 98.5 98.3

Table 5. Comparison of urinary cotinine levels (ng g)1 creatinine) among active smokers, peoplechewing nicotine gum, passive smokers, and non-smokers, using the two analytical methods

Method Smokers (n = 19) Substitute nicotinetherapyb (n = 7)

Passivesmokers (n = 7)

Non-smokers(n = 7)

P value

HPLC 3056.2 ± 2893.3a 2657.6 ± 2345.2 456.3 ± 356.2 96.2 ± 38.2 <0.05c

CE 2976.4 ± 2645.3 2367.2 ± 2178.8 496.8 ± 412.3 89.3 ± 24.5 <0.05

a Mean concentrations ± SDb Nicorette gumc One-way ANOVA test


comparative evaluation of ce and hplc for determination of cotinine in human urine

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