Journal of Chromatographic Science, Vol.

46, September 2008

Determination of Phenazopyridine in Human Plasma

by GC–MS and its Pharmacokinetics
Kai-jun Li1, Qin-hua Chen1,*, Zhuo Zhang2, Peng Zhou3, Peng Li1, Jia Liu2, and Jun Zhu1
1Department of Pharmacy, Affiliated Dongfeng General Hospital, Yunyang Medical College, Hubei 442008 PR China; 2School of Medicine,

Xi’an Jiaotong University, Xi’an 710061, PR China; and 3Longzhu Hospital of Shenzhen, Guangdong, 518026, PR China

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Abstract a great deal of plasma sample (1 mL) and extract (5 mL) were
necessary (10).
A sensitive, selective, and simple gas chromatography–mass This paper reports that a simple, sensitive, and specific gas
spectrometry method is developed for quantitation of chromatography (GC)–MS method was investigated for the
phenazopyridine (PAP) in human plasma using internal standard quantitation of PAP in human plasma samples with liquid–liquid
(diazepam). PAP and IS are extracted from plasma by liquid–liquid extraction as the sample preparation method for further
extraction and analyzed on a DB-5MS column with mass selective detection based on the GC–MS method for rats (11). We have
detector. Excellent linearity is found between 5–500 ng/mL (r = applied this method for bioequivalence study of two oral dosage
0.9992, n = 7) for PAP in human plasma. The limit of detection is forms of PAP (test and reference). The open randomized, cross
0.3 ng/mL. Intra- and Inter-day precisions expressed as the relative over study performed on a group of 18 healthy, Chinese male
standard deviation for the method are 1.37–6.69% and volunteers confirmed the bioequivalence of both formulations. It
1.24–6.01%, respectively. Extraction efficiency is more than 90%,
has proven that the method can be widely applied in phenazopy-
and recoveries are in the range of 92.65–96.21%. This method is
successfully applied for the pharmacokinetics and bioequivalence
ridine clinical practice, such as bioequivalence and bioavailablity
of 2 formulations of PAP in 18 healthy male volunteers who study.
received a single 200 mg dose of each formulation.
Experimental
Introduction
Chemicals and Reagents
Phenazopyridine hydrochloride exerts an analgesic effect on Phenazopyridine hydrochloride (purity > 99.0%) reference
the mucosa of the urinary tract and is used to provide symp- and reference tablets were supplied by Jia-lun Pharmaceutical
tomatic relief of pain in conditions such as cystitis and urethritis Company of Hong Kong. Test tablets were supplied by Institute
(1). It is given in conjunction with an antibacterial agent for the of Pharmaceutical Research of Gui-zhou. Diazepam (purity >
treatment of urinary-tract infections (2,3). 98.8%) was bought by National Institute for the Pharmaceutical
Phenazopyridine (PAP) [2,6-diamino-3-(phenylazo)pyridine] and Biological Products of China. HPLC grade methanol, ethyl
(Figure 1A) was adopted by USP in 1928 (4). Many methods have acetate, and diethyl ether were obtained from Fisher Scientific
been developed for the measurement of PAP such as a polaro- (Pittsburg, PA). Helium (purity, 99.999%) was from Xi’an
graphic and voltammetric study of copper-phenazopyridine Analytical Instrument Factory (Xi’an, China). Other reagents
monohydrochloride system in phosphate buffer medium elec- used in the experiment were analytical grade and from commer-
troanalysis (5), adsorptive stripping voltammetric assay of cial sources.
phenazopyridine hydrochloride in biological fluids and pharma-
ceutical preparations (6) and determination of phenazopyridine GC–MS conditions and instrumentation
in tablets by high-performance liquid chromatography (HPLC) A capillary GC/MS-QP2010 (Shimadzu, Kyoto, Japan) with a
(7,8), have been reported. However, these methods all have dis-
advantages for determination of biological matrices. LC and
LC–mass spectrometry (MS) methods for determination of PAP
in human plasma have been reported (9,10). The HPLC–UV
method was developed to determine phenazopyridine concentra-
tions in plasma at 405 nm, but the limit of quantitation (LOQ)
was as high as 50 ng/mL. Some treatment procedures given
showed that the extraction recoveries were lower than 80% and
Figure 1. The structures of phenazopyridine and diazepam.
* Author to whom correspondence should be addressed: email cqh77@163.com.

686 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
Journal of Chromatographic Science, Vol. 46, September 2008

DB-5MS capillary column (30 m × 0.32 mm i.d, 0.25 µm film the standard curve was prepared. Calibration curves were
thickness, Agilent Technologies, Palo Alto, CA) was used. The inlet obtained from plasma samples spiked with various amounts of
temperature was maintained at 280°C. The oven temperature was PAP (5, 10, 20, 50, 100, 200, and 500 ng/mL) and 50 µL of
initially held at 140°C for 2 min, and then programmed to 280°C working IS solution was added for all quantitated samples to
at 10°C/min where it was held constant for 4 min. Helium was evaluate the linearity, precision (%CV, n = 5), and accuracy (the
used as carrier gas at a constant flow rate of 2.0 mL/min. The bio- bias between theoretical and actual concentrations).
logical samples were analyzed by GC–MS with the pulsed splitless The intra-day and inter-day precision and accuracy were deter-
injection mode. 1 µL was injected for each assay. mined by analyzing spiked plasma samples containing 5, 20, 100,
The source and electrodes of the quadrupole mass filter were and 500 ng/mL of PAP from 5 analyses per day for 5 consecutive
both set to 200°C. Ionization was carried out in electron impact days, respectively. Recoveries of PAP and IS were calculated by
ionization (EI) mode at 70 eV. Detection was operated under comparing the peak areas of 5 extracted samples from plasma to
selected ion monitoring (SIM) mode. The ions for base peak (m/z that of standards injected directly at the concentrations of 5, 20,
213 for PAP and m/z 283 for IS) were selected for quantitative 100, and 500 ng/mL with the mean peak area of standards and
assay under investigation. 5.3 µg/mL of IS.

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Preparation of analytical standard solutions, Sample preparation
PAP quantitation, method validation A liquid–liquid extraction method was used for the extraction
The stock solution of PHP and IS were prepared by dissolving of PAP and IS in bio-samples. To every plasma sample, 50 µL of IS
PAP and I.S in methanol to make a 1.0 mg/mL solution, (µg/mL) was added to 0.5 mL plasma in glass centrifuge tube.
respectively. The regression line of peak-area ratios versus stan- Then, every solution was subjected to addition of 0.1 mL of 1M
dard concentrations calculated with respect to IS was used in NaOH at room temperature. Next, 2.5 mL ethyl acetate–diethyl
quantitation of PAP. As well as 5.3 µg/mL working solution of IS, ether (1:1) was added to every one, and the mixture was vortexed
the working standard solution for each concentration point on for 5 min. After centrifugation for 10 min at 4000 rpm, the upper
organic layer was transferred to a clean tube. The organic solution
was evaporated under a stream of nitrogen at 40°C. 0.1 milliliters
of ethyl acetate was added to the residue and centrifugation pro-
cedure mentioned above was repeated. An aliquot (1 µL) of the
supernatant was injected into the GC–MS systems for analysis.

Results and Discussion

Pharmacokinetic studies
PAP was given to healthy male volunteers after having
obtained their informed consents before enrolment to partici-
pate to the study. The clinical protocol was reviewed and
approved by the Ethics Committee of Xi’an Jiaotong University
Hospital, China. Eighteen healthy male volunteers aged from 21
to 28 and with body weight between 58 and 70 kg were included
in a randomized, single-dose, 2-period, 2-sequence, cross-over
Figure 2.Total ion current chromatogram of phenazopyridine and diazepam study with a 1-week washout period. The volunteers were non-
(A), mass-spectrogram of phenazopyridine (B), and mass-spectrogram of alcoholic, free from cardiac, hepatic, renal, gastrointestinal, and
diazepam (C).
hematological diseases, and also assessed by physical examina-
tion and following laboratory tests, had
complete blood count, hepatic function, and
Table I. Precision and Accuracy of the GC–MS Method for Determining PAP renal function tests. During each period, the
Concentration in Human Plasma Samples volunteers were admitted to the Clinical
Pharmacokinetic Laboratory, Xi’an Jiaotong
Intra-day (n = 5) Inter-day (n = 5)
Concentration University Hospital at 18:00 h and had an
added CF* Precision Accuracy CF Precision Accuracy evening meal before 20:00 h. After an
(ng/mL) (mean ± SD, ng/mL) (%) (%) (mean ± SD, ng/mL) (%) (%) overnight fasting, they received a single 200
mg PAP dose of each of the formulations of
5 5.160 ± 0.345 6.69 103.2 5.090 ± 0.279 5.48 101.8 test and reference drug at 07:00 h along
20 20.48 ± 0.444 2.17 102.4 19.90 ± 1.196 6.01 99.5
with 240 mL of water. They were then in the
100 94.50 ± 3.374 3.57 94.5 93.20 ± 2.488 2.67 93.2
500 499.0 ± 6.836 1.37 99.8 483.5 ± 5.995 1.24 96.7
seated position for at least 1 h and then
fasted for 2 h. A standard lunch and an
* CF = Concentration found. evening meal were provided at 4 and 10 h
after dosing. Blood samples (5 mL) were

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 Journal of Chromatographic Science, Vol. 46, September 2008

withdrawn from antecuvital vein at 0, 0.25, 0.50, 0.75, 1.0, 1.5, 2, The GC–SIM chromatograms obtained for blank samples, PAP,
3, 4, 6, 8, 12, and 24 h after and then transferred to vacutainer and IS are shown in Figure 3 as analyzed by GC–MS. The pres-
tubes and centrifuged at 4000 rpm for 15 min (4°C); the plasma ence of specific fragmentations, such as m/z of 213 and 283 are
samples obtained were stored at –20°C until analysis. The con- shown in Figure 2. It is obvious that the GC–SIM method sim-
centrations of total plasma of PAP were determined as the mean plifies the chromatogram very efficiently and provides a single
of duplicate samples. The maximal concentration (Cmax) and the peak for identification.
time for maximal concentration (Tmax) were determined by
visual inspection from each volunteer’s plasma concentra- Method validation and linearity of calibration curve
tion–time plots for PAP. The area under the plasma concentra- The peak area of PAP in human plasma was linear with respect
tion–time curve [AUC(0 – t)] was calculated by the linear to the analyte concentration over the range of (5–500 ng/mL).
trapezoidal method from 0 to 24 h. The AUC extrapolated to The mean linear regression equation of calibration for PAP was
infinity [AUC(0 – ∞)] was calculated as AUC(0 – t) + Ct/Ke, where Ct y = (0.03 ± 0.011) + (0.021 ± 0.001), r = 0.9992, n =7, where y
is the last measurable concentration and the elimination rate was the peak area of the analyte and x was the concentration of
constant (Ke) was obtained from the least square fitted terminal the analyte. LOQ was established as (0.3 ng/mL) for PAP with an

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log-linear portion of the plasma-concentration–time profile (The accuracy of 104% and precision less than 12%. The information
Pharmacological Society of China, 1997). Plasma elimination of precision and accuracy are shown in Table I. For an intra-day
half-life (T1/2) was calculated as 0.693/Ke. run (n = 5), the CVs of PAP at 5, 20, 100, and 500 ng/mL were
6.69%, 2.17%, 3.57%, and 1.37%, respectively. The accuracy
Separation which mean the bias between theoretical and actual concentra-
A typical gas chromatography-total ion current (GC-TIC) mass tions at the 5, 20, 100, and 500 ng/mL were 93.2–103.2%. The
spectrogram chromatogram obtained for PAP standard and IS is extraction recoveries of PAP and IS from spiked human plasma
shown in Figure 2 as analyzed by GC–MS in the electron impact (EI) were determined at the concentrations of 5, 20, 100, and 500
mode. The retention time of PAP was 12.5 min and IS was 13.8 min. ng/mL and 106 ng/mL in five replicates as shown in Table II. The
It is obvious that the GC–TIC chromatogram and mass spectrogram recoveries of PAP and IS were 91.89% and 93.21%, respectively (12).
can provide the elective ion and the time program: From 3–13.2 min
the m/z was 213 (PAP) and 13.2–20 min the m/z was 283 (I.S). Stability
Table III lists data for bench top, freeze/thaw, and storage sta-
Table II. The Extraction Recovery of Measurement of bility. Bench top stability was investigated to ensure that PAP was
PAP and IS in Human Plasma
Sample concentration Extraction recovery
Samples (ng/mL) (mean ± SD, n = 5)

Phenazopyridine 5 95.34 ± 6.59

20 93.34 ± 3.49
100 92.65 ± 4.21
500 96.21 ± 3.57
Diazepam 106 93.21 ± 2.48

Table III. Stability of PAP in Human Plasma

Nominal Mean found
concentration concentration Precision Accuracy
Figure 3. Selected ion monitoring chromatograms of blank plasma (A) and an
(ng/mL) (n = 5) (mean ± SD, ng/mL) (%) (%) extract of blank plasma (B) spiked with phenazopyridine (100 ng/mL) and
diazepam (106 ng/mL).
Bench 5 5.195 ± 0.379 7.3 103.9
top 20 20.48 ± 1.024 5.0 102.4
stability* 100 99.90 ± 2.697 2.7 99.9
500 493.0 ± 5.916 1.2 98.6
Freeze and 5 5.125 ± 0.384 7.5 102.5
thaw 20 20.26 ± 1.256 6.2 101.3
stability† 100 99.60 ± 2.789 2.8 99.6
500 509.0 ± 4.581 0.9 101.8
4-week 5 5.120 ± 0.353 6.9 102.4
storage 20 20.04 ± 1.042 5.2 100.2
stability‡ 100 101.3 ± 2.431 2.4 101.3 Figure 4. Mean concentration–time curve of phenazopyridine for two tablets
500 486.0 ± 5.832 1.2 97.2 in 18 healthy male volunteers after single oral administration of 200 mg.
* Exposed at room temperature (25°C) for 15 h.
Mean ± SD. Test drug and reference drug represented 200 mg tablets were
† After three freeze-thaw cycles. supplied by Institute of Pharmaceutical Research of Gui-zhou and Jia-lun
‡ Stored at −20°C. Pharmaceutical Company of Hong Kong, respectively.

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Journal of Chromatographic Science, Vol. 46, September 2008

Table IV. Mean Pharmacokinetic Parameters Obtained

Conclusions
from 18 Human Volunteers After Oral Administration of
200 mg of PAP Tablets A simple, sensitive and specific GC–MS method was
investigated for the quantification of PAP in plasma samples with
Pharmacokinetic Test drug Reference drug
liquid–liquid extraction as sample pretreatment. Diazepam was
parameters Mean ± S.D Mean ± S.D
selected as internal standard (IS, Figure 1B). The limit of detec-
AUC(0 – 12 h) (ng.h/mL) 354.15 ± 92.04 345.67 ± 81.52 tion of PAP was up to 0.1 ng/mL (S/N > 3) in biological samples.
AUC(0 – ∞) (ng.h/mL) 421.61 ± 77.29 431.77 ± 87.82 The whole procedure is sensitive, accurate, and reproducible,
Cmax (ng/mL) 62.76 ± 30.68 65.00 ± 29.23 which should be suitable to support this study. The applicability
Tmax (h) 2.65 ± 0.38 2.48 ± 0.50 of the method is demonstrated in the bioequivalence test of two
T1/2 (h) 9.61 ± 1.97 9.41 ± 2.04 different PAP formulations in 18 healthy male volunteers.
Ke (h–1) 0.076 ± 0.018 0.076 ± 0.019

not degraded in plasma samples at room temperature for a time Acknowledgments

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period to cover the sample preparation. Four sets of plasma sam-
ples at concentrations of 5, 20, 100, and 500 ng/mL were left at This work was supported by the Natural Science Foundation of
room temperature for 12 h. The samples were then processed affiliated Dongfeng General Hospital (2007ZY039).
and analyzed. The results indicated that PAP was stable during
the exposure period.
Freeze-thaw stability was evaluated for PAP using QC samples. References
The QCs were exposed to three freeze-thaw cycles; each cycle
consisted of removing the QCs from the freezer, thawing them 1. Y. Yadollah, A. Jaber, and A.K. Mehdi. Solubilities of phenazopyri-
unassisted to room temperature, kept at room temperature for dine, propranolol, and methimazole in supercritical carbon
4 h and re-freezing at −20°C. The samples were processed along dioxide. J. Pharm. Biomed. Anal. 3: 181–87 (2003).
2. M.N. Helen, K. Mark, W.J. Ben, and B. James. Phenazopyridine-
with a standard curve and concentrations were determined. This induced hemolytic anemia. Urology 21: 623–24 (1983).
result indicated that PAP had an acceptable stability after three 3. L. Jaime, K. Elizabeth, and L. Robert. Acquired methemo-
freeze-thaw cycles in human plasma. The storage stability at globinemia possibly related to phenazopyridine in a woman with
−20°C was also tested using QC samples. normal renal function. Urology 158: 1520–21 (1997).
The stability was closely monitored during validation and 4. B.T. Eleuterio, M.P. Yolanda, S. Martha, R. Morales, and J.N. Pedro.
1H, 13C and 15N NMR assignments of phenazopyridine deriva-
sample analysis periods, and no degradation of the compound was tives. Magnetic Resonance in Chemistry 43: 256–60 (2005).
observed. The 4-week stability data are listed in Table III. The 5. Ç. Osman and B. Ender. A polarographic and voltammetric study of
result indicated that PAP was stable in plasma for at least 6 weeks. the copper-phenazopyridine monohydrochloride system in phos-
phate buffer medium. Pharmacoepidemiology and Drug Safety 9:
Clinical application in healthy volunteers 87–90 (1997).
This method has been successfully used to the pharmacoki- 6. M. Suzy and Sabry. Adsorptive stripping voltammetric assay of
phenazopyridine hydrochloride in biological fluids and pharma-
netic study of PAP after a single oral dosing of two PAP formula- ceutical preparations. Talanta 50: 133–40 (1999).
tions (200 mg) for 18 healthy male volunteers by Latin-square 7. L. Jan, S. Du Preez, Alta Botha, Antonie P. Lötter. High-performance
cross-over design. Figure 4 shows the mean plasma concentra- liquid chromatographic determination of phenazopyridine
tion profiles of two formulations of PAP in 18 volunteers. hydrochloride, tetracycline hydrochloride and sulphamethizole in
Pharmacokinetic parameters are shown in Table IV. These combination. J. Chromatogr. A 333: 249–52 (1985).
8. F. Belal. Simultaneous high-performance liquid chromatographic
parameters of 2 formulations such as AUC, Cmax, Tmax, and T1/2 determination of phenazopyridine and nitrofurantoin in tablets.
are comparable to the corresponding parameters obtained by Chromatographia 25: 61–63 (1988).
single oral dose of 200 mg PAP in the results of Zhou Y.D. et al. 9. D. Farin, G. Piva, and R.K. Cohen. Determination of phenazopyri-
(13). In the bioequivalence test, the maximum concentration dine in human plasma by high-performance liquid chromatog-
(Cmax) and the areas under the curve [AUC(0 – 12 h)] were com- raphy. Chromatographia 52: 179–80 (2000).
10. E Shang, B. Xiang, G.Y. Liu, W.A. Wei, and L. Lu. Determination of
pared Zhou Y.D. et al. The geometric mean and 90% confidence phenazopyridine in human plasma via LC-MS and subsequent
interval (CI) of Cmax and AUC(0 – 12 h) were 100.5% (90% CI = development of a pharmacokinetic model. Anal. Bioanal. Chem.
92.42–117.99) and 100.9% (90% CI = 90.57–110.13), respec- 382: 216–22 (2005).
tively. Because both 90% CI for Cmax and AUC(0 – 12 h) were 11. Qinhua Chen, Kaijun Li, Zhuo Zhang, et al. Development and val-
included in the 80–125% interval proposed by the China Food idation of a gas chromatography–mass spectrometry method for the
determination of phenazopyridine in rat plasma: Application to the
and Drug Administration, it could be concluded that two types of pharmacokinetic study. Biopharm. Drug Dispos. 28: 439–44
200 mg tablets were considered to be bioequivalent according to (2007).
pharmacokinetic results of PAP obtained from time–concentra- 12. I. Shargel and A.B.C. Yu. Applied Biopharmaceutics and Pharma-
tion profiles of the two formulations. Diazepam, as the internal cokinetics. Appleton and Lange 193 (1993).
standard, is commonly used in clinical practice, so it may 13. Y.D. Zhou, H.X. He, and F. Qiu. Bioequivalence of phenazopyri-
dine hydrochloride. J. physio-Sci of SiChuan 26: 4 (2004).
interfere with the assay results of phenazopyridine. Therefore, a
full range mass scan was performed in volunteers’ blank plasma, Manuscript received August 28, 2007;
and no drug of benzodiazepines was found. revision received November 23, 2007.

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