Repetitive Determination of lsonicotinic Acid Hydrazide in Flow-Through Systems by Series Reactions V. V. S. Eswara Dutt and Horacio A. Mottola” Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74074
Microgram amounts of isonicotlnic acld hydrazide (lsonlarld) have been determlned on a repetltlve basis by sample InJectlon Into a continuously recirculated reagent reservoir solution. Chemically, the method makes use of two competltlve reactions: (a) formation, in phosphoric acld medium, of a colored complex between isoniazid and vanadlum(V), (b) destructlve oxldation of thls complex by excess of the V03- reagent. The rates of these two reactlons are adjusted to obtaln a transient slgnai whose height is directly proportional to the amount of Isoniazid In the injected sample.
Increased concern with health-related problems and with environmental changes and effects have generated renewed interest in the analytical chemistry of materials in both liquid and gaseous mixtures. A large portion of this interest is focused on meeting the challenges of automation whereby, at low cost, a large number of samples (rendering statistical treatment of data meaningful) can be processed with suitable precision and accuracy. Sample injection in flow-through systems offers several advantages in the area of continuous-flow chemical analysis. The main advantages are elimination of air segmentation, large number of determinations per unit of time, and simplification of flow-through schemes. Recently reported contributions to repetitive determinations in flow-through systems with sample injection address themselves to these points (1-3). In addition, the ancillary concepts of main reagent (or background solution) recirculation and regeneration are easily adapted to flow injection procedures (4-6). The work reported here is an application of sample injection in flow-through systems with reagent recirculation as well as of chemical determinations based on series reactions and/or processes (3,5). It deals with the determination of microgram amounts of isonicotinic acid hydrazide (isoniazid) by monitoring a transiently generated signal whose height is directly proportional to the amount of isoniazid in the injected sample. Isoniazid is commonly employed in the treatment of tuberculosis (7). Several methods have been reported for the determination of isoniazid in pharmaceutical formulations and in biological fluids, including titrimetry (8),spectrophotometry (9,l o ) , fluorometry (11), potentiometry (12), polarography (13), coulometry (14), atomic absorption (15),and radioimmunoassay (16). Blake, Bode, and Rhodes (17) compared four widely used standard methods (aqueous iodometric, bromometric, and nitrite titrations, and nonaqueous acid-base titration). They recommend the nitrite method on the basis of the recoveries obtained in presence of glucose, sucrose, and lactose. All reported methods have disadvantages such as reagent instability, need of multireagent solutions, and the requirement of laborious and time-consuming manipulations. Consequently the sampling rate is low, approximately 10 or less samples per hour. The novel procedure presented in this paper involves the use of a single reagent reservoir solution, is highly selective, and provides a high number of determinations per unit time (around 360 samples per hour). 776
ANALYTICAL CHEMISTRY, VOL. 49,
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EXPERIMENTAL Apparatus. The flow-through cell-reservoir combination assembly (Figure l),the sample injection unit, and the modular spectrophotometricunit used in these studies have been described in detail previously (3, 5). Reagents. The isoniazid used was from Aldrich Chem. Co., Inc., Milwaukee, Wis., and the isoniazid stock solutions were standardized titrimetrically with vanadate (8). All other reagents used were of analytical grade. Singly distilled water was found satisfactory for solution preparations and dilutions. Pharmaceutical formulations were 100-mgisoniazid tablets supplied by Oklahoma State University Hospital. Procedure for the Repetitive Determination of Isoniazid. The reagent reservoir solution was prepared in batches of 250 mL containing 50.0 mL of 0.0400 M ammonium vanadate solution, 40.0 mL of 5.00 M phosphoric acid, and 100 mL of water. The solution was circulated at a flow rate of 70 mL/min while continuous stirring was maintained both in the reservoir flask and in the photometric cell. A sample volume of 0.30 mL was injected by means of the sample injection system and the transient signal (Figure 1) was recorded in a Sargent SRG strip chart recorder. The amount of isoniazid was evaluated by reference to a working curve (Figure2) obtained with standards. The monitored complex between isoniazid and vmadium(V) shows maximum absorption at 420 nm. Procedure for the Determination of Isoniazid in Pharmaceutical Tablets. A previously weighed 100-mg isoniazid tablet was powdered in a mortar. The resulting material was weighed and dissolved in 100 mL of distilled water and the solution filtered through Whatman filter paper No. 42. The filtrate was finally diluted to 500 mL and 0.30-mL aliquots of this solution were used for injection. The content of isoniazid in the tablet was estimated with reference to the working curve and also by a standard addition procedure. RESULTS AND DISCUSSION The determination reported in this paper involves a fast complexation reaction in the detection zone (Equation 1) Isoniazid + V(V) --L Amber-colored Complex (11 followed by a slower redox process (Equation 2): Amber Complex + V(V) +. Isonicotinic Acid + N, + V(1V) (2) Adjusting the rates at which these consecutive processes take place so that: results in the peak signal profiles illustrated in Figure 1. The chemical reaction represented by Equation 1 was proposed by Rao et al. for the photometric determination of isoniazid (8). They assumed the formation of a 1:l complex with maximum absorption at -420 nm. Fast return to “baseline” conditions, as required for the determination of a large number of samples per unit time, demands a reasonably fast destruction of the amber-colored complex. At room temperature and relatively low excess of vanadate ion [the source of V(V)], the amber complex decomposes very slowly. Transient signals of analytical value can be obtained with the help of osmium(VII1) to catalyze the decomposition (5). If vanadate ion is in the order of M and phosphoric acid is used to provide
;,,
0
Absorhancc
I
1
C D
Figure 3. Signal profiles in different acid media obtained at zero flow rate. NaV03 0.0020M, isoniazid 1.50 X M. A 0.50 M H,PO,; B: 0.50 M HCI; C: 0.50 M HC104; D: Q.50M “03; E: 0.50M HPSOl
I
i i
j
1‘ L
Figure 1. (1) Flow-through cell and flow-through loop for repetitive, continuous injection determinations. (a)Teflon needle for sample injection connected to the force-flow injection system described in Figure 1 of (b) spectrophotometer cell spin-bar (Bel-Art Products, PeRef. (3); quannock, N.J.); (c)peristaltic pump (Masterflexwith SRC Model 7020 speed controller and 7014 pump head); (4 reagent solution reservoir, (e) magnetic stirring bar. (2)Typical signal profiles for isoniazid determination according to procedure described in text. Amount of isoniazid injected in pg: A, 57.6,,; B, 41.14;C, 32.9,;D, 24.6,; E, 16.4& F, 8.23
t
/
t
[YO;]
/([VO&] t [ISCWAZID])
Figure 4. Continuous variation data for the formation of the isonlazid-vanadium(V) complex in 1.OO M H3P04, A = 420 nm. Maximum occurs at 0.495
‘‘O[ I .D
1’
I ID.0
I
moo
I 10.0
I
I
I
IO.0
100
LOO
ISONIAZID. +g
Figure 2. Working curve for the determination of isoniazid according to procedure described in text. 1 cm = 0.038 absorbance unit
the needed hydrogen ion concentration, return to the baseline is sufficiently fast even in absence of catalyst. The need for phosphoric acid is notable, since signal heights are substantially lower in other acid media and the decomposition step is also slower. This is illustrated in Figure 3 showing signal profiles obtained at zero flow rate to ascertain the relative effect of the different acids employed. When hydrochloric-or perchloric acid is used, at optimum conditions
for repetitive determinations, as much as 15 min is needed for restoration of the curve to “baseline”; with phosphoric acid, under comparable conditions, the same restoration occurs in 10 s. Because a 1.00 M phosphoric acid provides the best medium for isoniazid determination, we investigated the complex formation in this particular medium. Continuous variation studies (Figure 4) confirm the isoniazid/vanadium(V) ratio of 1 in the complex with maximum absorption at 420 nm. Photometric titration of a constant amount of isoniazid with vanadate ion, however, reveals a molar absorptivity of 4746. This value is more than six times the value reported by Krych and Lipiec for the complex with nicotinic acid hydrazide in Robinson and Britton buffers (18). The value of the molar absorptivity and the continuous variation plot permit evaluating K = [Amber Complex]/ [Isoniazid][V(V)]. The calculated value for log K is 2.96 in 1.00 M phosphoric acid; in Robinson-Britton buffers of pH 1.5-2.7, Krych and Liepic reported a value of 2.14 for the log K for the V(V)nicotinic acid hydrazide complex (18). ANALYTICAL CHEMISTRY, VOL. 49,NO. 6, MAY 1977
777
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a
I
I
I 0.50
I
1
I
1.10
PHOSPHORIC A C I D
I
1
1.10
I
I
I NeVOs.
4
Figure 5. Effect of phosphoric acid concentration on peak height and signal duration at zero flow rate. Isoniazid 1.10 X 1O4 M; NaVO, 0.010 M; 1 cm = 0.038 A
I
I
I I&
1.6
0.4
I
m ll0'
Figure 6. Effect of vanadate concentration on peak height and signal M; H3P04 1-00 M; duration at zero flow rate. Isoniazid 1.10 X 1 cm = 0.038 A
In the presence of large excess of vanadate ion, complex formation is first order with respect to isoniazid and the complex decomposition is first order with respect to complex concentration. Consequently the following rate expression describes the transient signal: d [ complex] = k , ' [Isoniazid] - k,' [Complex] dt
(3)
4 4
This assumes pseudo-zero order dependence on vanadate ion and phosphoric acid, which are present in large concentration. Kinetic studies have shown that k( = 2.8 X lo3 s-' and k i = 2.8 X 10-1 s-'. The ratio k l ' / k i is thus -10000 and the theoretical recovery of "equilibrium signal" (signal value in absence of reaction 2) should be close to 100% (3). A 16% signal loss, however, was observed in practice and it is attributed to the flow contribution and mechanical carry-over which considerablyincrease the observed value of ki. Several other factors affect the signal profile for isoniazid determination and are discussed in detail below. Effect of Phosphoric Acid Concentration. A summary of the observations collected at zero flow rate and varying the H3P04concentration appears in Figure 5. This figure shows that phosphoric acid has a twofold effect: (a) the peak height decreases gradually with increasing acid concentration; this effect is less pronounced up to 1.50 M but becomes significant above 1.50 M H3P04,and (b) the signal duration dramatically decreases with increasing acid concentration up to 1.50-2.00 M H3P04;above 1.50 M H3P04,the effect is of little analytical significance. These effects are the combined result of H3P04 affecting the values of k{ and 122' through possible complex formation. Although a H3P04 concentrationof 1.50 M appears optimum according to Figure 5, a concentration of 1.00 M is recommended for isoniazid determination because the increased viscosity of the stronger acid introduces optical artifacts producing noisy signals. Effect of Sodium Vanadate Concentration. The effect of vanadate concentration on signal height and signal duration is shown in Figure 6. In view of the necessary compromise between large signal height and short signal duration, a vanadate concentration of 0.010 M is recommended. When the reagent reservoir mixture is prepared, at least 10 min should be allowed before the first injection. This time is needed for attainment of equilibrium in the formation of vanadate acid, which absorbs slightly at 420 nm. Effect of Temperature. Variation of temperature between 25.0 and 40.0 "C had no significant effect on signal height or signal duration. Signal height, for example, only increases -2% as the temperature is changed from 25.0 to 40.0 "C.
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
n
a
-10
10
40
F'.OW
60
'a
100
110
2
140
R A T E , rnlimln
Figure 7. Effect of flow rate on peak height and signal duration. Isoniazid 1.10 X lo4 M;H3P041.00 M NaVO, 0.010M 1 am = 0.038 A
Effect of Flow Rate and Sample Volume. Flow rate is a critical variable because of its effect on k i at relatively high values. Figure 7 illustrates the effect of flow rate on both signal height and signal duration. Larger values of k i can be obtained with increasing flow rates. This results in a shorter time for return to the baseline and in an increase in the number of determinations possible per unit time. Since the flow does not affect the value of k;, increasing flow rates yields decreasing values of k { / k d , which results in a decrease in signal height (3). When these effects are taken into account, observation of Figure 7 suggests a flow rate of 70 mL/min as the best compromise. Variation of the injected sample volume between 0.08 and 0.80 mL (keeping constant the fmd concentration of isoniazid) produced no significant effect on the signal profile. The recommended range, however, is 0.10 to 0.50 mL since larger volumes more rapidly decrease the usefulness of the reservoir solution because of dilution, and smaller volumes give poorer precision. Determination Rate, Precision, and Reservoir Capacity. The average duration of the transient signal dictates the number of injections that can be made per unit time. Under the conditions described in the Experimental section of this paper, return to baseline occurs in about 10 s and the estimated determination rate is 360 samples/h. The mean percent standard deviation for 20 coneecutive injections of 0.30-mL samples, each containing 25 Mg of isoniazid, was found to be 1.48%. The number of determinations that can be accomplished with a fixed volume of reservoir solution was estimated by repeated injection of the same volume of
Table I. Determination of Isoniazid in Pharmaceutical Tablets by the Proposed Sample Injection Procedure and the USP XVIII Methods. Nominal Content/Tablet: 100 mg
Tablet
A B
C
Sample injection InterpoStandard lation= additionu USP XVIII* 90.5 91.7 94.0
92.8 94.0 92.0
93.0 95.0 90.0
Reported values are average of 6 determinations. Reported values are average of 4 determinations.
a
standard isoniazid solution. A 5% loss of original signal height was observed with a 250-mL reservoir solution, containing 0.0100 M vanadate and 1.00 M phosphoric acid, after 400 repetitive injections of 0.30-mL samples containing 40 pg of isoniazid. The 5% signal loss is of no significance when the standard addition method is used. The number of samples that can be processed with a fixed volume of reservoir solution depends, of course, on the volume injected (dilution effect) and the absolute amount of isoniazid present in the sample (effective decrease of vanadate concentration). The high number of samples that can be processed also indicates that the accumulated products of the reaction do not interfere with the determination procedure. The nitrogen liberated in each injection creates no problems since the amount is very small and the bubbles are carried out by the flow of reagent to the reservoir, where most of them collapse. A debubbler located just before the flow-cell(6)helps to avoid noise due to bubble carryover at high flow rates. Interferences and t h e Determination of Isoniazid in Pharmaceutical Tablets. Common diluents in pharmaceutical preparations such as glucose, starch, citric acid, oxalic acid, fructose, and dextrin did not interfere with the isoniazid determination even if present in a thousandfold excess. Analysis of tablets was carried out as outlined under procedure by direct interpolation in the working curve (Figure 2) and
by standard addition. The results obtained are reported in Table I and compared with those obtained applying the USP XVIII procedure (19). The results obtained by the sample injection procedure compare well with the standard procedure. Direct interpolation of signal height into the working curve gave somewhat lower values than the standard addition procedure. The content of isoniazid found per tablet was, however, consistently lower than the minimum 98.0% stipulated by the USP XVIII (19).
ACKNOWLEDGMENT The assistance of Daniel Scheeler and William Janero in some of the experimental work reported here is gratefully acknowledged. LITERATURE CITED (1) J. RuiiEka and E. H. Hansen, Anal. Chim. Acta, 78, 145 (1975). (2) K. K. Stewart, G. R. Beecher, and P. E. Hare, Anal. Biochem., 70, 187 (1976). (3) V. V. S. Eswara Dutt, A. EskanderHanna,and H. A. Mottola, Anal. Chem., 48, 1207 (1976). (4) H. U. Bergmeyer and A. Hagen, Fresenius’ Z.Anal. Chem., 261, 333 (1972). (5) V. V. S.Eswara Dutt and H. A . Mottola, And. Chem., 47, 357 (1975). (6) V. V. S. Eswara Dutt and H. A . Mottola, Anal. Chem., 49, 319 (1977). (7) P. Dineen, in “Dnigs of Choice: 1972-1973”, W. Modeil, Ed., C. V. Mosby, St. Louis, Mo., 1972, pp 127-144. (8) P. V. K. Rao, G. B. B. Rao, and P. S. Murty, Mikrochim. Acta, 1974, 979. (9) H. S. I. Tan, J . Pharm. Sci., 62, 993 (1973). (10) J. T. Stewart and 0. A. Settle, J . Pharm. Sci., 84, 1403 (1975). (11) H. G. Boxenbaun and S. Riegeiman, J . Pharm. Scl., 63, 1191 (1974). (12) P. V. K. Rao and G. 8. B. Rao, Analyst(Lcndon), 96, 712 (1971). (13) J. J. Vallon, A. Eadinard, and C. Bichon, Anal. Chlm. Acta, 78, 93 (1975). (14) V. J. Jennings, A. Dodson, and A. Harrison, Analyst(London), e9, 145 (1974). (15) Y. Yoshinorl, K. Inagaki, T. Satome, and H. Koike, BunsekiKagaku, 22, 896 (1973). (16) R. Schwenk, K. Keiiey, K. S. Tse, and A. H. Sehon, Clin. Chem. ( Winston-Salem, N.C.), 21, 1059 (1975). (17) M. I. Blake, D. Bode, and H. J. Rhodes, J . Pharm. Sci., 83, 1303 (1974). (18) 2 . Krych and T. Lipiec, Chem. Anal. (Warsaw), 12, 535 (1967). (19) “The United States Pharmacopeia”, 16th rev., Ma& Publishing Co.,Easton, Pa., 1970, p 349.
RECEIVED for review December 22,1976. Accepted February 14,1977. This work was supported by the National Science Foundation (Grant CHE-76-03739).
Spectrophotometric and Stopped-Flow Reaction-Rate Study of the Formation of 1O-Molybdo-2-vanadophosphoric Acid Richard W. Hunt, Jr.’ and Larry G. Hargls” Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70 122
A compound widely used In the analysis of phosphate and vanadate was studled uslng spectrophotometrlc technlques and ldentlfled as 10-molybdo-2-vanadophosphoric acid (10M-2-VPA). Stolchlometry studies indicate that In the 0.3-1.5 pH range both Mo(V1) and V(V) exlst prlnclpally as dimers. Stopped-flow spectrophotometrlc reactlon-rate measurements were used to establlsh a rate law. Reactlon-rate data were acquired and manipulated with a mlnlcomputer controlled by an assembly-language program. The program facilitates averaging of multiple rate measurements, allows for lmmedlate vlsual display of the rate curve on a CRT, and uses a rapld least-squares sllgorlthm to determine lnitlal slopes of the reactlon-rate profiles.
‘Present address, TAN0 Corporation,4521 W. Napolean Avenue, Metairie, La. 70001.
Formation of a mixed vanadium-molybdenum heteropoly has been extremely widely used in analytical methods for determining phosphate (1-3). This rnixed complex appears to have two advantages over the use of the similar 12molybdophosphoric (12-MPA): a larger molar absorptivity for the mixed heteropoly generally leads to improved sensitivity ( I ) , and formation of the complex is less affected by commonly interfering ions (2,4). There has been considerable controversy over the appropriate solution conditions for an analysis (3-9) and the stoichiometry of the mixed heteropoly formed (1,4,6,10-13). There seems to be a general consensus that at low acidities yellow-orange molybdenum-vanadium complexes form and lead to unacceptable high-absorbing blanks, while at high acidities the rate of color formation is slow ( 4 , 6, 14). The nature of the mixed Mo-V isopoly complexes has not been satisfactorily studied and, although there is spectral and potentiometric evidence for the existence ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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