International Journal of Pharma and Bio Sciences

RESEARCH ARTICLE BIOPHARMACEUTICS

USE OF SIMPLE SPECTROPHOTOMETRIC METHOD FOR ESTIMATION OF

THEOPHYLLINE (TH) IN SALIVA AND URINE OF HEALTHY HUMAN
VOLUNTEER

VIKAS KUMAR2*, ARVIND SHARMA1, SANDEEP ARORA1 AND

VIPASHA DHILLON2
1
Chitkara College of Pharmacy, Chitkara University, Punjab
2
Chitkara School of Pharmaceutical Sciences, Chitkara University, Himachal Pradesh

VIKAS KUMAR
2
Chitkara School of Pharmaceutical Sciences, Chitkara University, Himachal Pradesh

*Corresponding author

ABSTRACT
A simple and sensitive spectrophotometric method was developed and validated for the
determination of Theophylline (TH) in pharmaceutical dosage form as well as in human
saliva and urine samples. This method is based on the measurement of absorbance of TH
in Phosphate Buffer 6.8 at 270 nm. Beer’s law is obeyed over the range of 2-10 µg/ml. This
method was used successfully for the quality assessment of Theophylline drug in human
urine and saliva samples with good precision and accuracy. The Sandell sensitivity, limit of
detection (LOD) and limit quantification (LOQ) values are also reported. All the methods
were validated in accordance with current ICH guidelines. The developed methods were
employed with high degree of precision and accuracy for the estimation of total drug
content in commercial tablet formulations of TH. The results obtained from human spiked
urine and saliva are satisfactory and recovery values were in limits.

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KEYWORDS
Absorbance, Buffer, Theophylline, UV anaylsis

INTRODUCTION
Systemic drugs administered orally or spectrophotometry. These methods require long
parenterally must reach the general circulation in and tedious pretreatment of the samples and
their pharmacologically active form to be laborious clean up procedures prior to analysis.
distributed throughout the body and to exert Therefore, it is very imperative to develop a
therapeutic effect. The intensity of the simple and suitable analytical method for the
therapeutic actions of many drugs correlate well determination of theophylline in saliva and urine.
with the concentration of the drug in the UV-Visible spectrophotometry is the technique of
biological fluid, Koch-weser et al (1982). The rate choice in research laboratories, hospitals and
of absorption is therapeutically important with pharmaceutical industries due to its low cost and
single doses of drugs, especially in case of inherent simplicity, Rajinder et al (2009).
narrow therapeutic index drugs, Benet et al
(1995). where relatively small changes in the Experimental Procedure
concentration can lead to marked changes in
pharmacodynamic response. Theophylline is a Materials
narrow therapeutic index drug and exhibits dose- Pure Theophylline was obtained as a gift sample
dependent pharmacokinetics, Dahlquist et al from Cipla Ltd. TH-1, TH-2 are Phylobid &
(1984). It is commonly used for the control and Theobid 200mg respectively. All the chemicals
management of acute asthma and acute were used of analytical grade. All Absorbance
exacerbations of chronic obstructive lung measurements were made on Shimadzu model
disease, Markham et al (1998). UV 1601 double beam UV Visible
The determination in biological fluids normally spectrophotometer with matched quartz
requires the use of trace analysis techniques cuvettes.
such as HPLC, LC, capillary electrophoresis
(CE), cyclic voltametry, LC-MS, gas Method
chromatography– mass spectrophotometry (GC- Calibration curve of Theophylline:
MS), inductively coupled plasma – mass

Fig - 1
Calibration curve of TH in phosphate buffer pH 6.8

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Aliquots of stock solution (50 µg/ml) were test. All the subjects abstained from any
pipette into a series of 10 ml volumetric flask xanthine-containing food or beverages or
and diluted to volume with phosphate buffer alcoholic products for 48 h prior to dosing and
pH (6.8). The reaction was allowed to proceed throughout the sampling schedule during each
at room temperature. The calibration curve period. Aliquot volumes of human urine
was constructed by plotting absorbance samples were transferred into small
against the initial concentration. The linearity separating funnel 10 ml of phosphate buffer
range or Beer’s range follows in between 2 to pH-6.8 were added and mixed well. A total of
10 µg/ml (Fig.1). The content of TH was 64 Urine samples (5 ml each) were collected
calculated either from the calibration curve or pre dose and at 0.5, 1.0, 2, 3, 4, 6, 8, and 10 h
corresponding regression equation and found post dose. The total volume of each interval
that the absorbance is stable for at least ten was recorded and a 10 ml sample each was
days at room temperature. Assay of tablet frozen until assay. Spectrophotometric
was done as per method prescribed in IP method was employed to determine the
(2007). elimination half life from the calibration graphs
or corresponding regression equation
Study design for urine and salivary
analysis Procedure for determination of
The study was conducted as an open label, theophylline in human saliva samples
balanced, randomized, two treatment, two Following overnight fast one tablet
period, two sequence single-dose crossover containing 200mg of Theophylline was given
study in 8 healthy adult, male, human orally along with 150 ml of water. The mouth
subjects, under fasting conditions, comparing was rinsed promptly with another 100 ml of
the concentration marketed formulation in water which was also swallowed. Food was
saliva and urine . Each subject received with held for a further period of 2 h to ensure
200mg theophylline (two different brands TH- complete absorption of the drug. Saliva
1,TH-2 of same drug) formulation with 240 ml samples were collected simultaneously at 0,
of drinking water after an overnight fast during 15, 30, 45, 60, 90, 120, 150, 180, 240, 300
each of the study day. There was a washout and 360 min. The salivary sample was
period of seven days between each study collected by placing citric acid (about 10 mg)
period for estimation of drug saliva and urine. over the tongue and held in the mouth for one
Informed consent was obtained from all to two minutes after which the contents were
subjects and the project was approved by the spit into a centrifuge tube. (Preliminary
Ethical Committee of our Institution (Chitkara experiments showed that citric acid did not
University). interfere with the estimation). Samples were
centrifuged to remove mucous and particulate
Procedure for determination of matter from saliva. The salivary supernatants
theophylline in human urine samples samples were stored at -20°C until analyzed.
Eight healthy adult male volunteers between The Theophylline concentration in saliva was
the ages of 21-25, weighing between 55 to 70 estimated by a modified method of Miceli et al
kg body-weight were selected for the study. (1979). For the determination of theophylline
Prior to initiation of the study the participants concentration in saliva: 1 ml of 20%
were subjected to thorough physical trichloracetic acid was added drop by drop to
examination and their medical history taken. 1 ml saliva with constant shaking. It was then
Basic tests like liver function test (LFT), urine centrifuged at 4000 rpm for 5 min followed by
analysis, full blood count (FBC) and blood 2000 rpm for 3 min. The supernatant (1.75 ml)
sugar levels were conducted for each subject, was transferred to a screw-capped 10 ml test-
to certify that they were medically fit for the tube followed by dilution with phosphate buffer

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(pH 6.8). The remaining procedure salivary absorbance. The linearity was evaluated by
estimation was the same as Miceli et al. linear regression analysis, which was
(1979). With the above modification calculated by the least square regression
concentration up to 1µg/ml could be detected method. The parameters LOD and LOQ were
in saliva. The salivary concentrations of determined on the basis of response and
theophylline were plotted against time on a slope of the regression equation. The
semi logarithmic graph paper and the accuracy and precision of the method was
elimination half life (t1/2%) was calculated. evaluated within the linear range based on the
Both protocol was in accordance to our analysis of TH samples and pharmaceutical
previous study a comparative bioequivalence formulations at 2.0, 4.0 and 6.0 µg/ml. Five
study of some brands of ofloxacin by urine independent analysis were performed at each
and salivary analysis in India, Sharma et al concentrations level within one day (Intra-day
(2010). precision) as well as for five consecutive days
(Inter-day precision). The accuracy was
RESULT AND DISCUSSION ascertained by recovery studies using the
standard addition method. The proposed
Method Validation method was used for estimation of TH from
The method was validated for selectivity, tablets after spiking with 50, 150 and 250 %
linearity, precision, accuracy, recovery and additional pure drug. The amount of TH was
stability according to the principles of the Food determined from the regression equation. The
and Drug Administration (FDA) industry absorbance-concentration plot for the
guidance. The specificity and selectivity of the proposed method was found to be rectilinear
proposed method was evaluated by over the range of 2- 10 µg/ml. Statistical
estimating the amount of TH in the presence analysis of regression lines were made
of common excipients. The linearity of the regarding the standard deviation of slopes
proposed method was constructed for TH (Sb) and standard deviation of intercepts (Sa)
standard stock solution by plotting and the values are summarized in Table 1.
concentration of the compound versus the

Table 1
Summary of optical and regression characteristics of the proposed method

Parameters Theophlline
Linear dynamic range (µg/ml) 2-20
Regression equation Y=4.5x10-2+0.157
Correlation coefficient (r) 0.998
LOD (µg/ml) 2.17x10-1
LOQ (µg/ml) 7.895x10-2

Estimation of drug in urine and saliva point of time are given in (Table.2). The plots
The proposed methods were found to be of average % cumulative of TH excreted over
simple, accurate and reproducible for routine a period of 12 h versus mid-point of time and
estimation of TH in urine and saliva. The average (log(dAU/dt )) versus mid-point of
average values for % cumulative amount of time, are shown in (Fig. 1& Fig. 2)
TH excreted, rate of excretion (dAU/dt ) and respectively. From these figures it is evident
log-transformed rate of excretion (log(dAU/dt that analysis of TH can be done for a period of
)) for both urine and saliva with respect to mid- 12 hrs in urine and saliva respectively.

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 Table 2
The average values for % cumulative amount of TH excreted, rate of excretion (dAU/dt ) and log-
transformed rate of excretion (log(dAU/dt ))
Mid Point of %age Cumulative Release Rate of Excretion log(dAU/dt ) (mg/h)
time (h) Urine Saliva Urine Saliva Urine Saliva
0.5 1.76 1.86 7.05 15.32 1.02 1.04
1 5.12 4.32 14.87 24.5 1.36 1.31
1.5 8.99 7.76 16.032 21.08 1.33 1.35
2 11.87 9.77 17.33 19.02 1.41 1.29
2.5 13.76 12.34 15.76 16.6 1.29 1.22
3 16.98 14.98 13.97 15.19 1.21 1.21
3.5 22.12 16.09 11.023 14.6 1.16 1.18
4 26.65 21.77 9.87 12.33 1.11 1.09
4.5 35.66 33.53 8.13 10.78 1.03 1.03
5 39.31 42.64 7.34 9.87 0.85 0.71
5.5 40.23 44.03 6.81 8.05 0.77 0.54
6 42.47 45.76 5.76 6.69 0.59 0.33

Fig.1
Average % cumulative TH excreted, versus mid-point of time plots after administration to
eight healthy male volunteers

Fig 2
Average log excretion rate (log (dAU/dt )) versus mid-point of time plots for TH after
administration to eight healthy male volunteers in urine and saliva

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Urinary excretion levels of TH after administration of saliva collected at different time intervals showed
Formulation TH-1&TH-2 (different brands) significant correlation (r = 0.64, P<0.05). We could
equivalent to 200mg of TH in eight volunteers was not find any significant difference between the
estimated using proposed spectrophotometric mean urine and salivary TH half lives.
method. The proposed method was used for
estimating of TH from tablets after spiking with 50,
150 and 250 % of additional pure drug. The CONCLUSIONS
selectivity of the proposed method was ascertained
by analyzing standard TH in the presence of The proposed method does not require any
excipients. It was observed that the excipients did laborious clean up procedure before measurement.
not interfere with the proposed method. The In addition, the method has wider linear dynamic
proposed method was further extended to the in range with good accuracy and precision. The
vitro determination of TH in spiked human urine method shows no interference from the common
samples.TH undergoes minimal metabolism in excipients and additives. This may help in
human with unchanged parent representing ≥90% analyzing affectivity of this drug in human beings
of drug derived in urine. This concentration fell well during treatment. Therefore, it is concluded that the
in within working range of proposed method. The proposed method is simple, sensitive and rapid for
calibration graphs were constructed by plotting the determination of in bulk, pharmaceutical
absorbance versus increasing concentrations of TH formulations and in human urine and salivary
in spiked human urine samples over the samples.
concentration range 2-10 µg/ml. The results
(Table.1) are satisfactorily accurate and precise. ACKNOWLEDGEMENT
The performance of the proposed method was
studied with other existing method, Gujral et al The authors wish to express their sincere thanks to
(2009). In case of proposed method and reported all the volunteers and Chitkara Educational Trust
method, the reported method has comparable LOD for their help in the earlier stage of this research
and LOQ value as in earlier reported method. work.
Analysis of TH concentration in 160 paired samples

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