Application of Liquid Chromatography to the Simultaneous Determination of Acetylsalicylic Acid, Caffeine, Codeine, Paracetamol, Pyridoxine, and Thiamine in Pharmaceutical Preparations

Abstract

This paper describes a rapid reversed-phase liquid chromatographic method, with UV detection, for the simultaneous determination of acetylsalicylic acid, caffeine, codeine, paracetamol, pyridoxine, and thiamine in pharmaceutical preparations. A reversed-phase C18 Nucleosil column is used. The mobile phase consists of 2 successive eluants: water (5 min) and acetonitrile-water (75 + 25, v/v; 9 min), both adjusted to pH 2.1 with phosphoric acid. Before determination acetylsalicylic acid is completely converted to salicylic acid by alkaline hydrolysis. Salicylic acid, caffeine, paracetamol, pyridoxine, and thiamine are all detected at 285 nm, whereas codeine is detected at 240 nm. Calibration curves were linear for salicylic acid, caffeine, paracetamol, and pyridoxine in the range of 50-500 mg/L, and for codeine and thiamine in the range of 50-1000 mg/L. The method was applied to the analysis of 13 fortified commercial pharmaceutical preparations. Recoveries ranged from 92.6 to 105.5%, with relative standard deviations of 1.1-5.8%.

Full text

DRUGS, COSMETICS, FORENSIC SCIENCES
Application of Liquid Chromatography to the Simultaneous
Determination of Acetylsalicylic Acid, Caffeine, Codeine,
Paracetamol, Pyridoxine, and Thiamine in Pharmaceutical
Preparations
NATIVIDAD RAMOS-MARTOS,FRANCISCO AGUIRRE-GÓMEZ, and ANTONIO MOLINA-DÍAZ
1
University of Jaén, Faculty of Experimental Sciences, Department of Physical and Analytical Chemistry, E-23071 Jaén,
Spain
LUIS F. CAPITÁN-VALLVEY
University of Granada, Faculty of Sciences, Department of Analytical Chemistry, E-18071 Granada, Spain
This paper describes a rapid reversed-phase liquid
chromatographic method, with UV detection, for
the simultaneous determination of acetylsalicylic
acid, caffeine, codeine, paracetamol, pyridoxine,
and thiamine in pharmaceutical preparations. A re-
versed-phase C
18
Nucleosil column is used. The
mobile phase consists of 2 successive eluants:
water (5 min) and acetonitrile–water (75 + 25, v/v;
9 min), both adjusted to pH 2.1 with phosphoric
acid. Before determination acetylsalicylic acid is
completely converted to salicylic acid by alkaline
hydrolysis. Salicylic acid, caffeine, paracetamol,
pyridoxine, and thiamine are all detected at
285 nm, whereas codeine is detected at 240 nm.
Calibration curves were linear for salicylic acid,
caffeine, paracetamol, and pyridoxine in the range
of 50–500 mg/L, and for codeine and thiamine in
the range of 50–1000 mg/L. The method was ap-
plied to the analysis of 13 fortified commercial
pharmaceutical preparations. Recoveries ranged
from 92.6 to 105.5%, with relative standard devia-
tions of 1.1–5.8%.
C
ombinations of analgesics as active principles in commer-
cial pharmaceutical preparations usually contain 2 or more
of the most common, i.e., acetylsalicylic acid (ASA),
salicylamide, paracetamol(PCT), andcodeine (CO),together with
central nervous system stimulants, e.g., caffeine (CF).
Gas chromatography (1, 2) and liquid chromatography
(LC; 3–5) have been used for the determination of these anal-
gesics. However, the LC methods developed for this purpose
deal usually with only 2 or 3 compounds (5–7). UV detection
is usually used (8–12), sometimes with precolumn
derivatization (13).
Some vitamins of the B-group, e.g., thiamine (TH) and
pyridoxine (PY), are found along with analgesics or central
nervous system stimulants in pharmaceutical preparations.
LC provides numerous methods for the separation and deter-
mination of vitamins of the B-group in different matrixes:
foods (14–16), medical foods (17), infant formula (18), and
pharmaceuticals (19, 20).
Nevertheless, only one method has been published for the
simultaneous determination of both water-soluble vitamins
and analgesics or central nervous system stimulants (9). The
method allows the determination of only 2 of the above men-
tioned analgesics and 1 vitamin B in a pharmaceutical prepa-
ration.
In this paper, we describe an LC method for the simulta-
neous determination of 6 active principles in pharmaceuticals.
Three of them are analgesics: ASA, CO, and PCT; 2 are wa-
ter-soluble vitamins: PY and TH; and one is a central nervous
system stimulant: CF.
The method described is sensitive, rapid, and reliable and
provides accurate results in analyses of pharmaceutical prepa-
rations.
Experimental
Reagents
(a) Stock solutions.—Stock solutions at 1.000 g/Lfor sali-
cylic acid (SA; Fluka, Madrid, Spain), CF (Panreac, Barce-
lona, Spain), PY hydrochloride (Fluka), and PCT (Fluka), and
at 10.000 g/L for CO (Abelló, Madrid, Spain) and TH hydro-
chloride (Fluka) were prepared by dissolution of the appropri-
ate amounts in water (LC grade). All chemicals were analyti-
cal grade, and ultrapure water was used. Stock solutions of
vitaminswere storedat 4°C. Working solutionswere prepared
daily by suitable dilution.
(b) Mobile phases.—Methanol (Fluka), acetonitrile
(Fluka), and ultrapure water were used. Phosphoric acid
(Panreac, Madrid, Spain) was added to adjust the pH of the
mobile phases after the acetonitrile and water were mixed.
Ultrapure water was obtained from a Milli-Q Plus system
(Millipore, Madrid, Spain; LC grade).
676 RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001
Received April 24, 2000. Accepted by JM July 20, 2000.
1
Author to whom correspondence should be addressed; e-mail:
amolina@ujaen.es.

RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 677
Table 1. LC parameters obtained under the operating conditions
a
of the developed method
Analyte
Retention time, t
R(min)
Capacity factor, k′
b
Asymmetry factor, SF
c
Resolution, R
s
d
Thiamine 4.297 0.480 3.2
7.93
Pyridoxine 7.085 1.438 2.6
19.01
Paracetamol 11.251 2.978 1.25
3.50
Codeine 11.690 3.315 1.9
3.70
Caffeine 12.150 3.301 3
10.74
Salicylic acid 13.363 3.375 2.64
a
Flow rate of mobile phase = 1 mL/min; column temperature = 35°C.
b
k′ =(t
R
–t
0
)/t
0
, where t
R
= retention time of each compound, and t
0
= retention time of the eluant (unretained compound).
c
SF = ratio of the 2 half-widths at 10% peak height.
d
R
s
=2)Z/(W
a
+W
b
), where )Z = distance between the maxima of 2 consecutive peaks, and W
a
and W
b
= peak widths.
Figure 1. Liquid chromatogram obtained by the developed method and showing the separation of the 6 compounds.
Conditions: detector 285 nm; concentration of each compound 200 mg/L. Peaks:1=TH;2=PY;3=PCT;4=CO;5=
CF; and 6 = SA.

678 RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001
Apparatus
(a) Liquid chromatograph.—Model HP 1050
(Hewlett-Packard Co., Arondale, PA).
(b) LC column.—Nucleosil C
18
stainless steel, 250 ×
4.6 mm, 5 µm particle size (No. 2515; Scharlau Science, Bar-
celona, Spain).
(c) Absorbance detector.—Hewlett-Packard diode-array
detector HP 1040 M series II with variable wavelength range
of 200–600 nm. Spectra of the eluates and absorbance mea-
surements at 285 and 240 nm were obtained at time intervals
of 0.640 s.
Treatment of Samples
The contents of capsules are quantitatively transferred, and
tablets are crushed to a fine powder for dissolution in water by
sonication. The solutions are then filtered through a 0.45 µm
pore size Millipore filter, andthe filtrates are diluted to appro-
priate volume with LC grade water. To obtain the complete
transformation of ASA to SA, alkaline hydrolysis is per-
formed for pharmaceuticals containing ASA by treating the
sample with 1M NaOH solution and heating at 60EC for
30min. Suitable dilutions aremadein all cases beforethesam-
ple solutions are injected.
LC Conditions
The following procedure is used for all samples: water
(pH 2.1) as the mobile phase for 5 min and then
acetonitrile–water (75 + 25, v/v) for 9 min, at a flow rate of
1 mL/min. The detection wavelength is set at 285 nm for SA,
CF, PCT, PY,and TH and at 240 nm forCO. The working col-
umn temperature is 35EC. The sample volume injected is
10 µL. The chromatographic parameters obtained under these
conditions are summarized in Table 1.
Table 2. Statistical parameters of calibration curves obtained for the developed method
Analyte Intercept Slope SD
a
a
SD
b
b
r
c
F
d
Salicylic acid –1.04 4.85 9.08 0.022 0.9998 1.27
Caffeine 59.08 11.84 25.54 0.091 0.9994 6.40
Codeine 89.48 6.15 19.34 0.037 0.9996 1.59
Paracetamol –5.29 4.41 9.08 0.032 0.9995 2.37
Pyridoxine –40.13 14.20 11.77 0.041 0.9999 2.23
Thiamine –8.37 2.66 4.38 0.008 0.9999 1.68
a
Standard deviation of the intercept.
b
Standard deviation of the slope.
c
Correlation coefficient.
d
F
ratio.
Table 3. Analytical parameters calculated for the developed method
Compound LDR, mg/L
a
RSD, %
b
DL, mg/L
c
Analyte concentration, 100 mg/L Analyte concentration, 400 mg/L Test I
d
Test II
e
Salicylic acid 50–500 1.65 1.47 27 5.1
Caffeine 50–500 5.84 2.84 13 4.9
Pyridoxine 50–500 2.12 1.87 27 10.5
Thiamine 50–1000 3.24 4.21
f
19 7.6
Codeine 50–1000 1.11 1.25
f
20 7.7
Paracetamol 50–500 3.51 2.33 17 6.6
a
LDR = linear dynamic range.
b
RSD = relative standard deviation.
c
DL = detection limit.
d
From ref. 21.
e
From ref. 22: DL =
S
b
2
1
y/x
n
n
−
−
; b = slope; and S
y/x
= standard deviation of y-residuals.
f
Analyte concentration = 800 mg/L.

LC Procedure
A10µL aliquot of solution containing the analytes in their
linear dynamic concentration ranges are injected into the liquid
chromatograph: 50–500 mg/L for SA, CF, PCT, and PY and
50–1000 mg/L for CO and TH. A flow rate of 1 mL/min and
working column temperature of 35EC are used. The com-
pounds are separated on a reversed-phase C
18
Nucleosil col-
umn, with a mobile phase consisting of the 2 successive eluants
described above, both adjusted to pH 2.1 with phosphoric acid
before the water and acetonitrile are mixed. After all the com-
pounds are separated, the water is passed through the column
for 4 min. Absorbance peak areas are measured in all cases.
Results and Discussion
Temperature and Flow Rate
In the proposed LC method, the temperature variation and
the flow rate for the resolution of the system did not have any
significant influence on theanalytical signal. However, a tem-
perature of 35EC was used because it allowed a liquid phase
with a lower viscosity, and a flow rate of 1 mL/min, which
was appropriate for the working pressure of the chromato-
graphic equipment, was used to shorten the time required to
perform the analysis.
Order of Elution
Preliminary studies with several eluant systems were con-
ducted to select the most effective eluant for the separation of
the 6 analytes of the system. With some eluants, water at
pH 5.0 and acetonitrile–water from (25 + 75, v/v) to (75 + 25,
v/v),also at pH 5,noresolution was observed because of large
overlap of the signals (for example, the CO and PY peaks).
Moreover, asymmetric and very wide peaks were obtained for
water-soluble vitamins.
With a more acidic eluant, acetonitrile–water (75 + 25,
v/v), pH 2.1, thinner peaks as well as a good separation were
achieved for CO with respect to water-soluble vitamins, but
PY and TH were not satisfactorily separated. With only water
at pH 2.1, PY and TH were separated satisfactorily, but the
other 4 compounds, with lower polarity, were not eluted. It
could be expected that the use of a less polar mobile phase af-
ter the elution of the water-soluble vitamins would separate
them. Methanol–water (35 + 65, v/v) was tried, but it failed to
produce a good separation for CO and PCT.
Finally, acetonitrile–water (75 + 25, v/v), pH 2.1, gave a
satisfactoryresolutionof the 4 compounds.Therefore,themo-
bile phase selected for the most efficient separation consisted
of 2 eluants: the first eluant was water (LC)adjusted to pH 2.1
with phosphoric acid. It allowed the separation of the vita-
mins, which were retained more weakly in the apolar station-
ary phase because oftheir ionic nature. The second eluantwas
acetonitrile–water (75 + 25, v/v),also adjusted to the same pH
with phosphoric acid, for the separation of the 4 remaining
components of the system. When the elution time for the first
eluant was increased from 3 to 5 min, the signal from PY was
greatly improved, producing only 1 peak (elution times of
<5min gave2 close peaks).This variable, however,had no in-
RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 679
Table 4. Recovery
a
of salicylic acid, caffeine, pyridoxine, thiamine, codeine, and paracetamol from synthetic mixtures
Level of each
analyte
added, mg/L
Salicylic acid Caffeine Pyridoxine Thiamine Codeine Paracetamol
Analyte
found, mg/L
Avg. rec. ±
SD, %
b
Analyte
found, mg/L
Avg. rec. ±
SD, %
Analyte
found, mg/L
Avg. rec. ±
SD, %
Analyte
found, mg/L
Avg. rec. ±
SD, %
Analyte
found, mg/L
Avg. rec. ±
SD, %
Analyte
found, mg/L
Avg. rec. ±
SD, %
50 50.0 100 ± 2 46.5 93 ± 1 50.3 100.6 ± 0.3 49.3 96 ± 2 45.1 90 ± 4 46.7 93.5 ± 0.1
200 199.8 99.9 ± 0.5 205.4 102.7 ± 0.2 198.9 99 ± 1 395.7
c
99 ± 2 402.7
c
100.7 ± 0.3 201.6 101 ± 3
500 500.4 100.1 ± 0.2 494.4 98.9 ± 0.1 500.7 100.2 ± 0.7 1001.4
d
100.3 ± 0.3 988.2
d
99 ± 1 498.2 99.7 ± 0.7
a
Each value is the mean of 3 replicate determinations.
b
SD = standard deviation.
c
Analyte added at 400 mg/L.
d
Analyte added at 1000 mg/L.

fluence on the resolution of the other compounds, including
TH. With the second eluant, 9 min was shown to be appropri-
ate to obtain satisfactory resolution (Table 1). A typical
chromatogram is shown in Figure 1.
Before the next injection the column was reequilibrated by
passing ultrapure water through the column for 4 min. This
was necessary to return the column to conditions appropriate
for separation of the water-soluble vitamins. Only after this
procedure were the peaks of the vitamins resolved.
Calibration and Analytical Parameters
Standard calibration graphs for the analytes were con-
structed by plotting peak areas produced by injection of stan-
dard solutions in the following concentration ranges:
50–500 mg/L for SA, CF, PCT, and PY,and 50–1000mg/L for
CO and TH. Calibration curves were analyzed by regression
analysis, and the correlation coefficient (r), slope, and
y-intercept for each run were calculated (Table 2). This process
was repeated 3 times for statistical analysis. The detection lim-
its (DL) for each compound, calculatedaccording to the criteria
of Miller and Miller (21) and Cuadros et al. (22), as well as the
linear dynamic ranges (LDR) and relative standard deviation
(RSD) at 2 analyte levels are shown in Table 3.
Applications
The proposed method was used in the simultaneous deter-
mination of the analytes in both synthetic mixtures and com-
mercial pharmaceutical formulations.
Synthetic mixtures.—Solutions containing the 6 active prin-
ciples at 3 different levels were prepared and analyzed by the
proposed method. Three determinations were performed in all
cases. Results found were completely satisfactory (Table 4).
Commercial pharmaceutical preparations.—Thirteen
commercial pharmaceuticals were analyzed by the developed
680 RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001
Table 5. Results
a
for the determination of acetylsalicyclic acid, caffeine, codeine, and paracetamol in commercial
pharmaceutical preparations
Pharmaceutical
preparation
Acetylsalicylic acid Caffeine Codeine Paracetamol
Label claim,
mg/unit
Avg. found ±
SD, mg/unit
b
Label claim,
mg/unit
Avg. found ±
SD, mg/unit
Label claim,
mg/unit
Avg. found ±
SD, mg/unit
Label claim,
mg/unit
Avg. found ±
SD, mg/unit
Analgilasa — — 30.0 27 ± 2 10.0 10 ± 1 500 458 ± 1
Dolmen 500 486 ± 3 — — 10.0 10.5 ± 0.2 — —
Dolvirán 400 387 ± 3 50.0 46 ± 1 9.6 9.4 ± 0.2 — —
Fiorinal 200 197 ± 3 40.0 31.1 ± 0.4 14.7 14.6 ± 0.2 300 274 ± 4
Rinomicine — — 30.0 27.7 ± 0.7 — — 150 138 ± 1
a
Each value is the mean of 3 replicate determinations.
b
SD = standard deviation.
Table 6. Results
a
for the determination of codeine, pyridoxine, and thiamine in commercial pharmaceutical
preparations
Pharmaceutical
preparation
Codeine Pyridoxine Thiamine (NO
3
–
or HCl)
Label claim,
mg/unit
Avg. found ± SD,
mg/unit
b
Label claim,
mg/unit
Avg. found ± SD,
mg/unit
Label claim,
mg/unit
Avg. found ± SD,
mg/unit
Benadom — — 300 277 ± 3 — —
Codeisán 28.7 26.1 ± 0.5 — — — —
Conductasa
c
30.7 29.9 ± 0.4 — — — —
Nervobión — — 100 103 ± 2 100 114.4 ± 0.3
Neurodavur — — 250 237 ± 6 250 241 ± 3
Pazbronquial 1.00 1.00 ± 0.01 0.60 0.6 ± 0.1 — —
Perduretas de codeína 50.0 46.1 ± 0.4 — — — —
Serfoxide
d
— — 300 280 ± 3 — —
a
Each value is the mean of 3 replicate determination.
b
SD = standard deviation.
c
Expressed as pyridoxine α-cetoglutarate.
d
Expressed as anhydre pyridoxine phosphoserinate.

RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 681
Table 7. Recovery
a
of salicylic acid, caffeine, pyridoxine, thiamine, codeine, and paracetamol from fortified commercial pharmaceutical preparations
Pharmaceutical
preparation
Salicylic acid Caffeine Pyridoxine Thiamine Codeine Paracetamol
Added, mg/L
Avg. rec. ±
SD, %
b
Added, mg/L
Avg. rec. ±
SD, % Added, mg/L
Avg. rec. ±
SD, % Added, mg/L
Avg. rec. ±
SD, % Added, mg/L
Avg. rec. ±
SD, % Added, mg/L
Avg. rec. ±
SD, %
Analgilasa 50 99 ± 1 100 101 ± 4 50 100 ± 2
100 105 ± 2 200 98 ± 4 200 98 ± 2
350 96.2 ± 0.2 300 98 ± 4 400 100 ± 1
Dolmen 20 98 ± 1 100 101 ± 1
50 99 ± 2 200 102 ± 1
100 99.4 ± 0.6 300 99.3 ± 0.7
Dovirán 50 101.4 ± 0.3 50 98 ± 1 100 99 ± 2
100 100 ± 1 100 101.7 ± 0.6 200 98 ± 2
150 99.3 ± 0.4 350 102.4 ± 0.5 300 99 ± 1
Fiorinal 50 98.4 ± 0.7 50 98 ± 1 100 98.4 ± 0.5
100 101.2 ± 0.3 200 99 ± 1 200 100 ± 1
150 101 ± 1 300 98.4 ± 0.6 300 102 ± 2
Nervobión 60 98.8 ± 0.2 60 99 ± 4
200 105 ± 2 200 105.5 ± 0.9
400 99 ± 2 800 99 ± 1
Neurovadur 60 101 ± 2 60 102 ± 5
200 100.8 ± 0.9 200 104 ± 2
400 99 ± 1 800 97 ± 3
Pazbronquial 100 92.6 ± 0.7 100 95 ± 1
200 101.1 ± 0.3 200 96 ± 1
300 104.6 ± 0.6 300 97 ± 1
Rinomicine 150 98 ± 1 400 95.5 ± 0.6
200 97 ± 2 800 96 ± 2
300 95 ± 2 1200 95 ± 2
a
Each value is the mean of 3 replicate determinations.
b
SD = standard deviation.

method after dissolution and suitable dilution as indicated in
the section on sample treatment. Results and label claims by
the manufacturer are summarized in Tables 5 and 6. In addi-
tion, as a check on the accuracy of the proposed method, a re-
covery study was performed in which the respective active
principles contained in several pharmaceutical preparations
were added at 3 levels. Good recoveries were obtained in all
cases (Table 7). Figure 2 shows the separation of the 4 com-
pounds (PCT, CO, CF, and SA) from Fiorinal capsules.
Conclusions
The proposed method allowed successful separation and
determination of 6 active principles: 1 organic acid (SA), 2 al-
kaloids (CF and CO), 2 water-soluble vitamins (PY and TH),
and a phenol derivative (PCT). The total time required for the
analysis is relatively shorter than that described by other re-
searchers (8, 9, 13) for simpler systems containing some of
these analytes (but not all). Its application to pharmaceuticals
after validation demonstrated that the proposed method can be
used satisfactorily for the determination of these analytes.
We compared the LC procedures proposed by the United
States Pharmacopeial Convention (USP; 23) with the devel-
oped method described here. The RSDs of the resultsobtained
by the USP methods and this method are similar; however, the
mobile phase of this method is simpler, and although the tail-
ingfactors,in general, are similar,theresolutionobtained with
this method is better. In addition, the method proposed in this
paper includes more compounds; the USP methods for CO
usually include another analgesic (such as PCT or ASA, but
not both simultaneously), and in no case do they include wa-
ter-soluble vitamins.
Acknowledgment
We acknowledge the financial support from the Ministerio
de Educación y Cultura de España (Dirección General de
Enseñanza Superior e Investigación Científica), project PB
97-0849.
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