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1071
ANALYST, JULY 1992, VOL. 117
Quantification of Theophylline in Human Plasma by Reversed-phase Ion-interaction High-performance Liquid Chromatography and Comparison With the TDx Fluorescence Polarization lmmunoassay Procedure
Downloaded by University of Guelph on 06 October 2012 Published on 01 January 1992 on http://pubs.rsc.org | doi:10.1039/AN9921701071
M. C. Gennaro, C. Abrigo and P. Biglino Dipartimento di Chimica Analitica, Universita di Torino, Via P. Giuria, 5-1-10 125 Torino, Italy
A new method for the determination of theophylline (1,3-dimethyl-l H-purine-2,6-dione) in human plasma is described, free from interference by theobromine (3,7-dimethyl-1 H-purine-2,6-dione) and caffeine (1,3,7tri met hyl-I H-pu rine-2,6-d ione). The method makes use of ion-i nteraction reversed-p hase high-performance liquid chromatography (octylamine-orthophosphate being the interaction reagent and a CI8 reversed-phase column the stationary phase) with spectrophotometric detection at 274 nm. The quantitative results obtained in the analysis of samples of plasma from patients undergoing treatment with theophylline were compared with those obtained for the same samples with the TDx fluorescence polarization immunoassay procedure (using the Abbot Therapeutic Drug Monitoring system), which is generally employed in hospitals and clinical laboratories. Statistical F-test and t-test for multiple samples were applied to the data obtained by the t w o methods. The results showed no significant difference between the t w o methods at the 95% confidence level. Keywords: High-performance liquid chromatography; theophylline; plasma; fluorescence polarization imm unoassay
Theophylline (1,3-dimethyI-lH-purine-2,6-dione) (see Fig. 1) acts as a stimulator of the central nervous system and is employed in medicine, particularly in the treatment of acute and chronic bronchial asthma and apnoea in premature newborns and infants.1-4 The therapeutic concentration of theophylline in the serum of adults ranges between 5 and 20 ppm and at concentrations higher than 20 ppm theophylline can become toxic.' Furthermore, it has been shown that the rate of clearance varies considerably among different individuals. For these reasons, during medical treatment with theophylline, in order to optimize the therapy doses1 it is necessary to monitor its concentration in plasma constantly. Gas chromatographic methods5.6 as well as reversed-phase high-performance liquid chromatography (HPLC)1,2,4,7-1" have been used for the determination of theophylline in biological specimens. A new HPLC method is proposed here, which makes use of reversed-phase ion-interaction chromatography, already employed in this laboratory in the development of analytical separation methods. 11-14
By employing octylamine-rthophosphate as the interaction reagent, a reversed-phase CI8 column as the stationary phase and spectrophotometric detection (at 274 nm), a method for the separation of xanthine (3,7-dihydro-lHpurine-2,6-dione), theobromine (3,7-dimethyl-lH-purine2,6-dione) , theophylline (1,3-dimethyl-lH-purine-2,6-dione), and caffeine (l73,7-trirnethyl-1H-purine-2,6-dione) was developed. The method was then employed in the quantitative analysis of theophylline in samples of human plasma obtained from patients undergoing therapy with theophylline. The results were compared, through statistical tests, with those obtained for the same samples with the so-called TDx method (using the Abbot Therapeutic Drug Monitoring system), which is a fluorescence polarization immunoassay procedure, commonly used in hospital and clinical laboratories .4
Experimental Apparatus
H
(343
1
2
Analyses were carried out with a Merck-Hitachi Lichrograph chromatograph Model L-6200, equipped with a two-channel Merck-Hitachi Model D-2500 Chromato-Integrator, interfaced with a UV-UVNIS detector L-4200. For pH measurements, a Metrohm 654 pH meter equipped with a combined glass-calomel electrode was employed and for the evaluation of absorptivity values a Hitachi 150-20 spectrophotometer was used. Chemicals and Reagents
3
4
Fig. 1 Structural formulae for: 1, xanthine (3,7-dihydro-lH-purine2, 2,6-dione), k, (40°C)= 1.19 X lo-'", k b (40°C)= 6.09 X theobromine (3,7-dimethyI-lH-purine-2,6-dione) k, (18 "C) = 0.90 X 10-10, kb 1.30 x 10-14; 3, theophylline (1,3-dimethyl-lH-purine-2,6dione), k, (25°C)= 1.69 x k b (25°C) = 1.90 X lo-14;and 4, caffeine (1,3,7-trimethyI-lH-purine-2,6-dione), k, (25"C) = 1.0 X k b = 0.70 x
Ultrapure water from a Millipore Milli-Q system was used for the preparation of solutions. Octylamine, orthophosphoric acid, xanthine, theophylline, theobromine and caffeine were Merck reagents and all other reagents were Fluka analyticalreagent grade chemicals. Chromatographic Conditions Merck Lichrospher 100 RP-18,5 pm, endcapped, packed in a 250 x 4 mm column was used as the stationary phase. Octylamine-rthophosphate 0.0050 mol dm-3 to be used as
View Online
Downloaded by University of Guelph on 06 October 2012 Published on 01 January 1992 on http://pubs.rsc.org | doi:10.1039/AN9921701071
1072
ANALYST, JULY 1992, VOL. 117
the interaction reagent was prepared by dissolving a weighed amount of octylamine in ultrapure water and by adjusting the pH value of the solution to 6.4 f 0.4 through the addition of orthophosphoric acid. The chromatographic system was conditioned by passing the eluent through the column until a stable baseline signal was obtained (a minimum of 1 h was necessary). The repeatability of measurements, with regard to both retention time and integrated absorbance, was 2% for sequential measurements (with the same conditions of eluent preparation and column conditioning) and the reproducibility (for different eluent preparations) was always within 6%. The eluent was freshly prepared each third day and the column was regenerated by passing ultrapure water (10 min, flow rate = 0.3 cm3 min-1) and then a water-methanol mixture 1 + 1 v/v (30 min, flow rate = 0.5 cm3 min-1) through it.
Preparation of Standard Solutions and Samples The standard solutions of theophylline and theobromine were prepared at concentrations of 500.0 ppm in dark-glass flasks and stored in a refrigerator at 4 "C. The samples of plasma to be analysed were prepared by centrifugation at 8000g for 15 min, dilution with ultrapure water (1 + 9 v/v) and filtration through a 0.20 pm Anotop disposable syringe filter.
Results and Discussion Previous workll-14 performed in this laboratory has been devoted to the development of separation methods by means of HPLC ion-interaction chromatography. The interaction reagent is prepared before the analysis from an acid and an amine (at a pH of 6.4 k 0.4, at which the acid is dissociated and the amine protonated). This represents the mobile phase, which, when flowing under isocratic conditions determines the so-called dynamic functionalization of the column, whose interaction properties can therefore be modified. This chromatographic system is suitable for separation studies of species that are able to give rise, under the working pH
conditions, to ion-pairs with the anion or with the protonated amine of the interaction reagent. Previous studies1*712 have shown that analytes are retained and then released as ion-pairs. Up to now inorganic and organic acids have been investigated,llJ2 in addition to amines,12J3 amides and imidesl4 characterized by suitable pK values. This paper shows also that xanthinic structures such as xanthine, theophylline, theobromine, and caffeine can be retained. However, by considering their structure and pK, and pKb values reported in the literature15 (see Fig. l), it cannot be clearly established which is the functional group responsible for the retention at the working pH value of 6.4. Conditions were investigated for the development of a separation method capable of allowing the determination of theophylline, free from interference from xanthine, caffeine and theobromine, which can also be present, under certain conditions, in human plasma. The separation obtained for a mixture of xanthine (2.00 pprn), theobromine (1.OO ppm) , theophylline (1 .OO ppm) and caffeine (3.00 ppm) is shown in Fig. 2. Spectrophotometric detection at 274 nm was chosen, at which wavelength all the species considered are characterized by high absorptivities, namely, E (theophylline) = (1.08 k 0.04) x 104, E (theobromine) = (0.99 k 0.05) x 104 and E (caffeine) = (0.96 _+ 0.05) x 104 dm3 mol-1 cm-1. The separation in Fig. 2 shows very good resolution, especially if one considers the very similar structures of the analytes and in particular the isomerism of theobromine and theophylline. The detection limit of theophylline, given as a signal-to-noise ratio of 3 : 1, was evaluated as 0.10 ppm. A standard graph was then constructed in the concentration range 0.10-3.00 pprn and good linearity [correlation coefficient ( r ) = 0.99891 was obtained between peak area and standard concentration. The method was then applied to the analysis of theophylline in human plasma. The method proved to be suitable because of a very low matrix interference at this wavelength, as shown in Fig. 3, which presents as an example a chromatogram recorded for a human plasma sample which is characterized by the absence of theophylline. Fig. 4, shows examples of
3.64
11.14
A 4 3
51.91 1.16
L 13.70
0
10
20
30
40
50
60
Time/mi n
Fig. 2 Separation of a mixture of: A , xanthine (2.0 ppm); B , theobromine (1.0 pprn), C, theophylline (1.0 pprn); and D, caffeine (3.0 pprn). Stationary phase: Merck Lichrospher 100 RP-18 (250 x 4 mm), 5 pm, endcapped. Ion-interaction reagent: 0.0050 mol dm-3 octylamine-orthophosphate. Flow-rate: 2.0 cm3 min-1, 100 mm3 injected. Spectrophotometric detection: 274 nm. Recorder setting: 0.002 a.u.f.s.
0
10
20
30 40 50 Ti me/min
60
Fig. 3 Chromatogram recorded for a sample of human plasma (diluted 1 + 9 v/v) which does not contain theophylline. Recorder setting, 0.002 a.u.f.s. Other conditions as in Fig. 2. Peak A, theobromine
View Online
1073
ANALYST, JULY 1992, VOL. 117
obtained by the TDx method. The average data obtained for the two series of measurements are given in Table 1. A graph reporting the results obtained with the HPLC method against those from the TDx method gave a correlation coefficient of 0.9921. The F-test was then performed for the two sets of data, whose degrees of freedom are v1 and v2, respectively. The calculated Fparameter ( F = sx2/s,2) was equal to 1.06. As the tabulated value of the F-distribution at the 95% confidence level and for v1 = v2 = 5 degrees of freedom was 5.05 it can be concluded that there is no significant difference between the variances of the two methods. The data presented in Table 1 were also treated with the so-called ‘t-test for multiple samples’. This test is particularly suitable for the analysis of different samples of slightly varying composition, as is usually the case with biological fluids in which the analyte is present in narrow physiological concentration ranges. According to this intercalibration test, the t value is evaluated through the parameter Di, computed as the difference between the results obtained with the two methods for the same sample with regard to the sign, as
lb) 2.84
4
Downloaded by University of Guelph on 06 October 2012 Published on 01 January 1992 on http://pubs.rsc.org | doi:10.1039/AN9921701071
2.86
,
0
I-
-
10
I
20
30
I
0
l
10
where
l
20 30 Ti me/m i n
0
10
20
30
Fig. 4 Chromatogram recorded for three samples of plasma (diluted 1 9 v/v), containing: ( a ) 2.05 ppm of theophylline; ( b ) 0.60 ppm of theophylline; and ( c ) 0.40 ppm of theophylline. Other conditions as in Fig. 2. Peaks: A , theobromine; and B , theophylline
+
Table 1 Comparison of results (ppm) obtained for different samples of plasma using the TDx and proposed HPLC methods. Average data from triplicate measurements are reported Sample A B C D E F
TDx method 4.02 6.87 18.60 20.39 20.50 20.89
Proposed HPLC method 3.91 6.23 18.80 18.20 20.70 19.42
chromatograms recorded for plasma obtained from three different patients who were undergoing theophylline-based therapy. These samples contain 2.05, 0.60 and 0.40 ppm of theophylline, respectively. In all of the chromatograms, besides the theophylline peak (peak B), two other major peaks can be observed; one of them (peak A) was identified as theobromine. Quantitative analysis of theophylline was performed at 274 nm, which corresponds to the wavelength of maximum sensitivity. The plot of peak area (y) versus concentration ( x ) , obtained by standard additions of theophylline to plasma samples showed a good linear trend ( y = -0.027 + 0 . 6 7 4 ~r; = 0.9841). The recovery of theophylline from spiked plasma was always greater than 98.8%. As mentioned, in hospital and clinical laboratories theophylline is generally determined through the TDx immunoassay procedure, based on measurements of polarized light fluoresence.4 In order to compare the results obtained from the proposed HPLC method with those from the TDx method, the analysis was performed using the two methods for a series of six samples of plasma obtained from six different patients treated with theophylline. Each analysis was repeated in triplicate. Repeatability was within 2% and reproducibility within 6%. Reproducibility data are comparable with those
D
is the mean of all the individual D idifferences and sd
=
dc(Di - D)2/(N - 1)
From the data in Table 1 a t value equal to 1.68 was calculated. The tabulated value of the t-distribution at the 95% confidence level and 5 degrees of freedom of 2.57 confirms that there is no significant difference between the two methods (at this confidence level). In conclusion, even if the results obtained with the HPLC method seem on the average slightly lower than those from the TDx method (see Table l ) , the F- and t-tests indicate that the methods are comparable at a significance level of 0.05. Reproducibility over time was also checked: results are reproducible for up to 2 d after which time the theophylline content appeared to be progressively lower. The methodology proposed here for the quantification of theophylline in plasma presents the advantage of being free from interference from xanthine, theobromine and caffeine and it does not require a pre-treatment step (apart from centrifugation and 0.20 pm filtration) or pre-column derivatization of the sample before injection. This work was supported by the Consiglio Nazionale delle Ricerche (CNR, Roma, Italia) and by the Minister0 della Ricerca Scientifica e Tecnologica (MURST, Roma, Italia).
References 1 Matsumoto, K., Kikuchi, H . , Iri, H . , Takahasi, H . , and Umino, M., J . Chromatogr., 1988, 425, 323. 2 Wahllander, A . , Renner, A . , and Karlaganis, G . , J. Chromatogr., 1985, 338, 369. 3 Lee, B . L., Jacob, P., and Benowitz, N . L., J. Chromatogr., 1989, 494, 109. 4 Blanchard, J . , Harvey, S . , and Morgan, W. J . , J. Chromatogr. Sci., 1990, 28, 303. 5 Argoudelis, C. J., J. Chromatogr., 1984, 303, 256. 6 Kasuya, Y . , Furuta, T., and Shimoto, H . , J . Chromatogr., 1989, 494, 101. 7 Wenk, M . , Eggs, B . , and Follath, F . , J . Chromatogr., 1983,276, 341. 8 Kinberger, B . , and Holmen, A . , J. Chromatogr., 1982, 229, 492. 9 Hartley, R . , Cookman, J . R . , and Smith, I . J., J . Chromatogr., 1984, 306, 19. 10 Naline, E . , Flouvat, B . , Advenier, C . , and Pays, M., J . Chromatogr., 1987,419, 177. 11 Gennaro, M. C., and Bertolo, P. L., J. Chromatogr., 1989,472, 433. 12 Gennaro, M. C., and Bertolo, P. L., J . Chromatogr., 1990,509, 147.
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13 Gennaro, M. C., Bertolo, P. L., and Marengo E., J. Chromatogr., 1990,518, 149. 14 Gennaro, M. C., and Abrigo, C . , J. Chromatogr. Sci., 1991,29, 410. 15 Smith, R . M., and Martell, A. E., Critical Stability Constants, Plenum Press, New York, London. 1976.
ANALYST, JULY 1992, VOL. 117 16 The Merck Index, eds. Budavari, S . , and O’Neil, M. J . , Merck Rahway, NJ, 1989.
Paper I/05316G Received October 21, I991 Accepted February I I , I992
1071
ANALYST, JULY 1992, VOL. 117
Quantification of Theophylline in Human Plasma by Reversed-phase Ion-interaction High-performance Liquid Chromatography and Comparison With the TDx Fluorescence Polarization lmmunoassay Procedure
Downloaded by University of Guelph on 06 October 2012 Published on 01 January 1992 on http://pubs.rsc.org | doi:10.1039/AN9921701071
M. C. Gennaro, C. Abrigo and P. Biglino Dipartimento di Chimica Analitica, Universita di Torino, Via P. Giuria, 5-1-10 125 Torino, Italy
A new method for the determination of theophylline (1,3-dimethyl-l H-purine-2,6-dione) in human plasma is described, free from interference by theobromine (3,7-dimethyl-1 H-purine-2,6-dione) and caffeine (1,3,7tri met hyl-I H-pu rine-2,6-d ione). The method makes use of ion-i nteraction reversed-p hase high-performance liquid chromatography (octylamine-orthophosphate being the interaction reagent and a CI8 reversed-phase column the stationary phase) with spectrophotometric detection at 274 nm. The quantitative results obtained in the analysis of samples of plasma from patients undergoing treatment with theophylline were compared with those obtained for the same samples with the TDx fluorescence polarization immunoassay procedure (using the Abbot Therapeutic Drug Monitoring system), which is generally employed in hospitals and clinical laboratories. Statistical F-test and t-test for multiple samples were applied to the data obtained by the t w o methods. The results showed no significant difference between the t w o methods at the 95% confidence level. Keywords: High-performance liquid chromatography; theophylline; plasma; fluorescence polarization imm unoassay
Theophylline (1,3-dimethyI-lH-purine-2,6-dione) (see Fig. 1) acts as a stimulator of the central nervous system and is employed in medicine, particularly in the treatment of acute and chronic bronchial asthma and apnoea in premature newborns and infants.1-4 The therapeutic concentration of theophylline in the serum of adults ranges between 5 and 20 ppm and at concentrations higher than 20 ppm theophylline can become toxic.' Furthermore, it has been shown that the rate of clearance varies considerably among different individuals. For these reasons, during medical treatment with theophylline, in order to optimize the therapy doses1 it is necessary to monitor its concentration in plasma constantly. Gas chromatographic methods5.6 as well as reversed-phase high-performance liquid chromatography (HPLC)1,2,4,7-1" have been used for the determination of theophylline in biological specimens. A new HPLC method is proposed here, which makes use of reversed-phase ion-interaction chromatography, already employed in this laboratory in the development of analytical separation methods. 11-14
By employing octylamine-rthophosphate as the interaction reagent, a reversed-phase CI8 column as the stationary phase and spectrophotometric detection (at 274 nm), a method for the separation of xanthine (3,7-dihydro-lHpurine-2,6-dione), theobromine (3,7-dimethyl-lH-purine2,6-dione) , theophylline (1,3-dimethyl-lH-purine-2,6-dione), and caffeine (l73,7-trirnethyl-1H-purine-2,6-dione) was developed. The method was then employed in the quantitative analysis of theophylline in samples of human plasma obtained from patients undergoing therapy with theophylline. The results were compared, through statistical tests, with those obtained for the same samples with the so-called TDx method (using the Abbot Therapeutic Drug Monitoring system), which is a fluorescence polarization immunoassay procedure, commonly used in hospital and clinical laboratories .4
Experimental Apparatus
H
(343
1
2
Analyses were carried out with a Merck-Hitachi Lichrograph chromatograph Model L-6200, equipped with a two-channel Merck-Hitachi Model D-2500 Chromato-Integrator, interfaced with a UV-UVNIS detector L-4200. For pH measurements, a Metrohm 654 pH meter equipped with a combined glass-calomel electrode was employed and for the evaluation of absorptivity values a Hitachi 150-20 spectrophotometer was used. Chemicals and Reagents
3
4
Fig. 1 Structural formulae for: 1, xanthine (3,7-dihydro-lH-purine2, 2,6-dione), k, (40°C)= 1.19 X lo-'", k b (40°C)= 6.09 X theobromine (3,7-dimethyI-lH-purine-2,6-dione) k, (18 "C) = 0.90 X 10-10, kb 1.30 x 10-14; 3, theophylline (1,3-dimethyl-lH-purine-2,6dione), k, (25°C)= 1.69 x k b (25°C) = 1.90 X lo-14;and 4, caffeine (1,3,7-trimethyI-lH-purine-2,6-dione), k, (25"C) = 1.0 X k b = 0.70 x
Ultrapure water from a Millipore Milli-Q system was used for the preparation of solutions. Octylamine, orthophosphoric acid, xanthine, theophylline, theobromine and caffeine were Merck reagents and all other reagents were Fluka analyticalreagent grade chemicals. Chromatographic Conditions Merck Lichrospher 100 RP-18,5 pm, endcapped, packed in a 250 x 4 mm column was used as the stationary phase. Octylamine-rthophosphate 0.0050 mol dm-3 to be used as
View Online
Downloaded by University of Guelph on 06 October 2012 Published on 01 January 1992 on http://pubs.rsc.org | doi:10.1039/AN9921701071
1072
ANALYST, JULY 1992, VOL. 117
the interaction reagent was prepared by dissolving a weighed amount of octylamine in ultrapure water and by adjusting the pH value of the solution to 6.4 f 0.4 through the addition of orthophosphoric acid. The chromatographic system was conditioned by passing the eluent through the column until a stable baseline signal was obtained (a minimum of 1 h was necessary). The repeatability of measurements, with regard to both retention time and integrated absorbance, was 2% for sequential measurements (with the same conditions of eluent preparation and column conditioning) and the reproducibility (for different eluent preparations) was always within 6%. The eluent was freshly prepared each third day and the column was regenerated by passing ultrapure water (10 min, flow rate = 0.3 cm3 min-1) and then a water-methanol mixture 1 + 1 v/v (30 min, flow rate = 0.5 cm3 min-1) through it.
Preparation of Standard Solutions and Samples The standard solutions of theophylline and theobromine were prepared at concentrations of 500.0 ppm in dark-glass flasks and stored in a refrigerator at 4 "C. The samples of plasma to be analysed were prepared by centrifugation at 8000g for 15 min, dilution with ultrapure water (1 + 9 v/v) and filtration through a 0.20 pm Anotop disposable syringe filter.
Results and Discussion Previous workll-14 performed in this laboratory has been devoted to the development of separation methods by means of HPLC ion-interaction chromatography. The interaction reagent is prepared before the analysis from an acid and an amine (at a pH of 6.4 k 0.4, at which the acid is dissociated and the amine protonated). This represents the mobile phase, which, when flowing under isocratic conditions determines the so-called dynamic functionalization of the column, whose interaction properties can therefore be modified. This chromatographic system is suitable for separation studies of species that are able to give rise, under the working pH
conditions, to ion-pairs with the anion or with the protonated amine of the interaction reagent. Previous studies1*712 have shown that analytes are retained and then released as ion-pairs. Up to now inorganic and organic acids have been investigated,llJ2 in addition to amines,12J3 amides and imidesl4 characterized by suitable pK values. This paper shows also that xanthinic structures such as xanthine, theophylline, theobromine, and caffeine can be retained. However, by considering their structure and pK, and pKb values reported in the literature15 (see Fig. l), it cannot be clearly established which is the functional group responsible for the retention at the working pH value of 6.4. Conditions were investigated for the development of a separation method capable of allowing the determination of theophylline, free from interference from xanthine, caffeine and theobromine, which can also be present, under certain conditions, in human plasma. The separation obtained for a mixture of xanthine (2.00 pprn), theobromine (1.OO ppm) , theophylline (1 .OO ppm) and caffeine (3.00 ppm) is shown in Fig. 2. Spectrophotometric detection at 274 nm was chosen, at which wavelength all the species considered are characterized by high absorptivities, namely, E (theophylline) = (1.08 k 0.04) x 104, E (theobromine) = (0.99 k 0.05) x 104 and E (caffeine) = (0.96 _+ 0.05) x 104 dm3 mol-1 cm-1. The separation in Fig. 2 shows very good resolution, especially if one considers the very similar structures of the analytes and in particular the isomerism of theobromine and theophylline. The detection limit of theophylline, given as a signal-to-noise ratio of 3 : 1, was evaluated as 0.10 ppm. A standard graph was then constructed in the concentration range 0.10-3.00 pprn and good linearity [correlation coefficient ( r ) = 0.99891 was obtained between peak area and standard concentration. The method was then applied to the analysis of theophylline in human plasma. The method proved to be suitable because of a very low matrix interference at this wavelength, as shown in Fig. 3, which presents as an example a chromatogram recorded for a human plasma sample which is characterized by the absence of theophylline. Fig. 4, shows examples of
3.64
11.14
A 4 3
51.91 1.16
L 13.70
0
10
20
30
40
50
60
Time/mi n
Fig. 2 Separation of a mixture of: A , xanthine (2.0 ppm); B , theobromine (1.0 pprn), C, theophylline (1.0 pprn); and D, caffeine (3.0 pprn). Stationary phase: Merck Lichrospher 100 RP-18 (250 x 4 mm), 5 pm, endcapped. Ion-interaction reagent: 0.0050 mol dm-3 octylamine-orthophosphate. Flow-rate: 2.0 cm3 min-1, 100 mm3 injected. Spectrophotometric detection: 274 nm. Recorder setting: 0.002 a.u.f.s.
0
10
20
30 40 50 Ti me/min
60
Fig. 3 Chromatogram recorded for a sample of human plasma (diluted 1 + 9 v/v) which does not contain theophylline. Recorder setting, 0.002 a.u.f.s. Other conditions as in Fig. 2. Peak A, theobromine
View Online
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ANALYST, JULY 1992, VOL. 117
obtained by the TDx method. The average data obtained for the two series of measurements are given in Table 1. A graph reporting the results obtained with the HPLC method against those from the TDx method gave a correlation coefficient of 0.9921. The F-test was then performed for the two sets of data, whose degrees of freedom are v1 and v2, respectively. The calculated Fparameter ( F = sx2/s,2) was equal to 1.06. As the tabulated value of the F-distribution at the 95% confidence level and for v1 = v2 = 5 degrees of freedom was 5.05 it can be concluded that there is no significant difference between the variances of the two methods. The data presented in Table 1 were also treated with the so-called ‘t-test for multiple samples’. This test is particularly suitable for the analysis of different samples of slightly varying composition, as is usually the case with biological fluids in which the analyte is present in narrow physiological concentration ranges. According to this intercalibration test, the t value is evaluated through the parameter Di, computed as the difference between the results obtained with the two methods for the same sample with regard to the sign, as
lb) 2.84
4
Downloaded by University of Guelph on 06 October 2012 Published on 01 January 1992 on http://pubs.rsc.org | doi:10.1039/AN9921701071
2.86
,
0
I-
-
10
I
20
30
I
0
l
10
where
l
20 30 Ti me/m i n
0
10
20
30
Fig. 4 Chromatogram recorded for three samples of plasma (diluted 1 9 v/v), containing: ( a ) 2.05 ppm of theophylline; ( b ) 0.60 ppm of theophylline; and ( c ) 0.40 ppm of theophylline. Other conditions as in Fig. 2. Peaks: A , theobromine; and B , theophylline
+
Table 1 Comparison of results (ppm) obtained for different samples of plasma using the TDx and proposed HPLC methods. Average data from triplicate measurements are reported Sample A B C D E F
TDx method 4.02 6.87 18.60 20.39 20.50 20.89
Proposed HPLC method 3.91 6.23 18.80 18.20 20.70 19.42
chromatograms recorded for plasma obtained from three different patients who were undergoing theophylline-based therapy. These samples contain 2.05, 0.60 and 0.40 ppm of theophylline, respectively. In all of the chromatograms, besides the theophylline peak (peak B), two other major peaks can be observed; one of them (peak A) was identified as theobromine. Quantitative analysis of theophylline was performed at 274 nm, which corresponds to the wavelength of maximum sensitivity. The plot of peak area (y) versus concentration ( x ) , obtained by standard additions of theophylline to plasma samples showed a good linear trend ( y = -0.027 + 0 . 6 7 4 ~r; = 0.9841). The recovery of theophylline from spiked plasma was always greater than 98.8%. As mentioned, in hospital and clinical laboratories theophylline is generally determined through the TDx immunoassay procedure, based on measurements of polarized light fluoresence.4 In order to compare the results obtained from the proposed HPLC method with those from the TDx method, the analysis was performed using the two methods for a series of six samples of plasma obtained from six different patients treated with theophylline. Each analysis was repeated in triplicate. Repeatability was within 2% and reproducibility within 6%. Reproducibility data are comparable with those
D
is the mean of all the individual D idifferences and sd
=
dc(Di - D)2/(N - 1)
From the data in Table 1 a t value equal to 1.68 was calculated. The tabulated value of the t-distribution at the 95% confidence level and 5 degrees of freedom of 2.57 confirms that there is no significant difference between the two methods (at this confidence level). In conclusion, even if the results obtained with the HPLC method seem on the average slightly lower than those from the TDx method (see Table l ) , the F- and t-tests indicate that the methods are comparable at a significance level of 0.05. Reproducibility over time was also checked: results are reproducible for up to 2 d after which time the theophylline content appeared to be progressively lower. The methodology proposed here for the quantification of theophylline in plasma presents the advantage of being free from interference from xanthine, theobromine and caffeine and it does not require a pre-treatment step (apart from centrifugation and 0.20 pm filtration) or pre-column derivatization of the sample before injection. This work was supported by the Consiglio Nazionale delle Ricerche (CNR, Roma, Italia) and by the Minister0 della Ricerca Scientifica e Tecnologica (MURST, Roma, Italia).
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ANALYST, JULY 1992, VOL. 117 16 The Merck Index, eds. Budavari, S . , and O’Neil, M. J . , Merck Rahway, NJ, 1989.
Paper I/05316G Received October 21, I991 Accepted February I I , I992
