application for a QCT as well as to future considerations of other, still latent, analytical uses.
and a K,,,FP (25) of 1.95 (determined concurrently with dextrose in water), an average M, value for NaCl of 29.76 f. 0.40 is obtained. The procedural simplification possible and the ease of operation with a QCT, in any circumstance requiring temperature data on a time basis, is remarkable. Furthermore, it appears that a QCT and digital recorder could be used to accumulate efficiently all the necessary temperature and time data from appropriate specimens, for application of the appropriate correction factors to establish the exact temperature at which an infinitesimal amount of the solid phase exists in equilibrium with liquid, in air at one atmosphere for an ultrapure substance. Primarily, by these examples, this report alerts analytical chemists to this new
This endeavor would not have been possible without the cooperation and support of numerous individuals in the Analytical and Spectroscopic Divisions of the Central Research Department, especially A. G. Bolinski for obtaining the mass spectra and Fulton G. Kitson for their interpretation; John W. Robson for benzene distillations; Philip B. Sweetser and Benjamin R. Stevens for G C analyses; and J. J. Skokowski for technical assistance. A. R. McGhie purified the DMSO by the normal freezing process.
(25) Ref. I, p 39.
RECEIVED for review July 9, 1971. Accepted November 3, 1971.
ACKNOWLEDGMENT
Simultaneous Automated Determination of Hydralazine Hydrochloride, Hydrochlorothiazide, and Reserpine in Single Tablet Formulations Tibor Urbanyi and Arthur O'Connell Analytical Research and Development Division, CIBA Pharmaceutical Company, Summit, N.J. An automated method for the simultaneous determination of three components present in a single tablet has been developed. Each tablet was individually analyzed for reserpine, hydralazine hydrochloride, and hydrochlorothiazide content at the rate of 10 tablets per hour. The reserpine content was assayed by a fluorometric procedure in the presence of the other components after the interference due to hydralazine hydrochloride was eliminated. The hydralazine hydrochloride component was determined by a new colorimetric method employing blue tetrazolium reagent. Hydrochlorothiazide was analyzed by ultraviolet absorption after removal of the interferences due to hydralazine hydrochloride and reserpine by an ion exchange column. The proposed method describes in detail the necessary chemical separations, optimum reaction conditions, and mechanical variables for each active ingredient.
THEPURPOSE of this study was to find a suitable method for the simultaneous, automated determination of a multicomponent pharamceutical formulation. The therapeutic formulation used in this study was an antihypertensive tablet containing three active ingredients ; reserpine, hydralazine hydrochloride, and hydrochlorothiazide. An extensive literature search revealed a large number of analytical methods available for each of the components. However, this does not mean that these methods can be applied for multicomponent determinations without any difficulties. The majority of the analytical methods for reserpine are based on colorimetric ( I ) or fluorometric ( 2 ) nitrite procedures. Recently a new, fluorometric method (3) using vanadium pentoxide for the oxidation of reserpine has been published, and applied successfully in the automated analysis (1) C. R. Szalkowski and W. J. Mader, J Amer. Pharm. Ass., Sei. Ed., 45, 613 (1956). (2) W. J. Mader, R. P. Haycock, P. E. Sheth, and R. J. Connelly, J. Ass. Ofic. Agr. Chem., 43,291 (1960). (3) T. Urbhnyi and H. Stober, J . Pharm. Sci., 59, 1824 (1970).
of reserpine tablets (4). This method was chosen, with some modifications, as a basis for the determination of reserpine in our formulation. Most methods for hydralazine hydrochloride are based on the reactive hydrazine group (5-7). Although these methods are suitable for single component determinations, difficulties occurred when hydralazine HCl is in the presence of other components. Potentiometric titrations in nonaqueous media with perchloric acid (8) and sodium methoxide (9) and aqueous titration with potassium bromate (10) are commonly used for hydralazine detection but these methods cannot be employed because of technical difficulties involved in automation. A colorimetric method using blue tetrazolium chloride was found to be satisfactory. Although polarographic (11) as well as titrimetric (12) determinations are known, spectrophotometric methods are the most common procedures for the hydrochlorothiazide detection. These methods can be classified into two groups; with and without hydrolysis of the parent compounds. Hydrolysis results in the formation of a disulfonamide and formaldehyde. Both compounds are detected colorimetrically, the disulfonamide after diazotization coupled with N-(lnaphthy1)ethylenediamine (13) or with chromotropic acid (4) T. Urbdnyi, and H. Stober, unpublished work. ( 5 ) R. Ruggieri, Farmaco, Ed. Prat., 11,571 (1956). (6) B. Wesley-Hadzija and F. Abaffy, Croat. Chem. Acta., 30, 15 ( 1958).
(7) G. I. Luk'yanchikova, Byul. Izobret., No. 14, 44 (1962). (8) R. Ruggieri, Bel/. Chim. Farm., 95, 382 (1956). (9) Ya. M. Perel'man and K. I. Eustratova, Aptech. Delo, 12, 45
(1963). (10) G. Cavicchi Sandri, Boll. Chim. Farm., 96,431 (1957). (11) A. I. Cohen, B. T. Keeler, N. H., Coy, and H. L. Yale, ANAL. CHEM.,34, 216 (1962). (12) P. Kertesz, Acta Pharm. Hung., 33, 150 (1963). (13) M. Ghelardoni and M. Fedi, Boll. Chim. Farm., 101,26 (1962).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
565
4mm.
6
hmm.
Table 1. Programming the SOLIDprep Sampler HomogeCycle nization Mixing Sampling Cleaning Dial setting M 8 48-63 63-82 82-100 Time in seconds 175 58 68 59 of full cycle 65 19 16
(14) and formaldehyde with the common reagent 2,4-dinitrophenylhydrazine (15). The diazotization method was successfully used for automated determination of hydrochlorothiazide (16). The nonhydrolyzed compound was determined by UV or IR (17,18) absorption measurements. The UV absorption method was employed successfully.
Figure 1. Schematic diagram of mixing device
(14) C. R. Rehm and J. B. Smith, J . Amer. Pharm. Ass., Sci. Ed., 49 396 (1960). (15) R. Fried, Mitt. Deut. Pharm. Ges., 34, 189 (1962). (16)K. B. Wrightman and W. W. Holl, Ann. N . Y . Acad. Sci., 130, 516 (1965). (17) H. Kala, Pharmuzie, 16, 297 (1961). (18) H. Marciszewski, Farm. Polska, 17,193 (1961).
DISCUSSION
150 mm long Solvaflex tubing and fill by drawing a water slurry of the resin through the tubing with vacuum. MIXER. An 80 X 3-mm six-chambered glass mixing tube was used instead of a regular mixing coil for the hydrochlorothiazide assay. The mixing tube has the dual adEXPERIMENTAL vantage of higher mixing efficiency and the elimination of a Reagents and Solutions. SATURATED SOLUTION OF VANADIUM pump tube. See Figure 1. CONTINUOUS FILTER.Remove the stirring rod from the PENTOXIDE.Filter through a medium porosity sintered plastic block to facilitate the flow through the filter. Use glass funnel a saturated solution of vanadium pentoxide in paper TO14 and set the speed so that the wet line is 6 to 7 mm phosphoric acid. in front of the lower block, HYDROGEN PEROXIDE 0.3 %. Dilute 1.0 ml of 30% hydroSOLIDprep SAMPLER.A 10-tablet per hour timing gear gen peroxide in 100 ml of water. This solution must be made was employed during this study. The slow speed motor confresh daily. trol was set to number 8. The homogenization, sampling and BLUETETRAZOLIUM REAGENT 0.02 %. Dissolve 760 mg of wash cycles were adjusted according to Table I. blue tetrazolium chloride in 3.8 liters of USP alcohol. Store AUTOMATED PROCEDURE. Assemble the automated systhe solution in an amber bottle. tem according to the flow diagram (Figure 2). This diagram TETRAMETHYLAMMONIUM HYDROXIDE 0.1 %. Dilute 38 indicates the automated equipment used for the analytical ml of 10 aqueous tetramethylammonium hydroxide with 3.8 procedure, and special tubings where necessary. Set the liters of USP alcohol. program on the SOLIDprep Sampler dial as indicated in Table Solvent. Prepare by mixing equal volumes of absolute I. In performing the analysis, tablets were placed individually methanol and water. One milliliter of concentrated phosinto the SOLIDprep Sampler turntable cups and the system phoric acid is added to each gallon of solvent. was activated. The tablets were ground to a fine powder Wash and Diluent Solutions. Prepare 50 % VjV methanol in in the blender and dissolved in 100 ml of solvent. After water. filtration of the insoluble tablet excipients from the soluStandard Solution. Weigh exactly 21.0 mg of reserpine tion, the flow from the filter was divided into three sample reference standard into a 200-ml volumetric flask; dissolve streams. The sample stream for the hydralazine hydrothe reserpine with absolute methanol, and dilute to volume. chloride determination was combined with the tetramethylAccurately weigh into a 100-ml volumetric flask 315 mg of ammoniumhydroxide and blue tetrazolium chloride reagent, hydrochlorothiazide and 525 mg of hydralazine hydrochlosegmented with air, and passed through a double mixing coil. ride reference standards. Pipet into this flask 20.0 ml of the Af,ter mixing, the solution was debubbled and pumped reserpine solution and dilute to volume with 50 % methanolthrough a 2-mm flow cell. The spectral measurement was water solvent. Prepare a fresh standard daily and keep it in made against air as reference using the automated slit control a dark place when not in use. and 530-nm wavelength on the spectrophotometer. The hyApparatus. A standard Technicon AutoAnalyzer system drochlorothiazide sample stream was pumped through an ion consisting of the following modules was used: SOLIDprep exchange column which bound the hydralazine hydrochloride Sampler programmed at 10 samples per hour; Proportioning as well as the reserpine but allowed the hydrochlorothiazide to Pump 11; Continuous Filter; two Beckman DB-G Spectropass through. This solution was mixed in a specially made photometers ; Beckman Hydrogen Lamp Power Supply ; two mixer and finally pumped through a IO-mm silica flow cell Technicon Recorders; Aminco SPF 125 Fluorometer; and a for measurement against air at 271 nm in an UV spectroSargent SRLG Recorder. Special Apparatus and Adjustments. ION EXCHANGE photometer. A separate sample line for reserpine determination was air segmented and mixed first with hydrogen peroxide COLUMN.Amberlyst 15 (Rohm and Haas) cation exchange and then with vanadium pentoxide reagent. The solution was resin is sieved through wire screens and the 40-60 mesh porpassed through a time delay coil for fluorescence development tion is placed into a 1-inch glass column. Isopropyl alcohol before measurement. The fluorometer was set at full sensiis passed through the resin until the effluent has no apprecitivity on the 30% range with an activation wavelength of able ultraviolet absorption in the 270-nm region; 1 N HC1 is 365 nm and an emission wavelength of 495 nm both with passed through the column to ensure total conversion of the 4-mm slit widths. A 2-mm flow cell was used. Five-milliliter resin to the acid form. Preparation of the resin is completed aliquots of the standards were introduced at the proper interby washing with water until the effluent is neutral to litmus. vals during the measurements. From the curves, the content Place a small glass wool plug in one end of a 1 mm i.d. by per tablet was calculated.
566
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
When Content Uniformity Testing became official in the Compendia (See US.Pharmacopeia), its inclusion and function was two-fold; to assure the consumers throughout the country of the quality of the content uniformity of tablets and
Name of Product(s1:
Dote :
SERaAP-ES
April 13,1~71
Automated Method (s):
Fluorometric, Colorimetric, Ultraviolet
Operator:
Type of Sompla:
Tablet
Affiliation: CIBA Pbrm.
Concentration of Active Ingredient(s 1 per Unit Dose:
A. OConnell
0.1:25:15 mg
Tube Size
SOLID prep SAMPLER CONTINUOUS FILTER Rate o f Sample ....I! per hour.
AO
Dissolution:
.!.? mi of .!E. ...,
hhlax 530 nrn 2 rnrn all
Methanol
DO
o
s
t x
Legend: Polyethylene Solvaflex Standard Teflon
hAcriv: 385 nm
h Fluor: 485 nrn FLUOROMETER
PROPOR TI ON1 NG PUMP
Figure 2. Flow diagram for Ser-Ap-Ek tablets to provide the manufacturer with the official requirements and technical data for good manufacturing practice. This new regulation was more than a matter of academic interest. T o fulfill the requirements and guarantee the safety and efficacy of the drug used in tablets, the pharmaceutical companies faced not only economical problems but also technical difficulties. Automated analysis is increasingly used to overcome these problems and to benefit manufacturers as well as to provide the consumer with added safeguards. The product used for this study was an antihypertensive pharmaceutical formulation with the trade name of “SerAp-Es.” “Ser-Ap-Es” is an abbreviation derived from its three active ingredients ; Serpasil [reserpine], Apresoline [hydralazine HCl], and Esidrix [hydrochlorothiazide] in 0.1 :25 :15 mg concentrations, respectively. The problem was to peform all three analyses simultaneously on a single tablet. The analytical methods were performed manually in preliminary studies in order to establish the optimum operating conditions for the automated system. The rate at which tablets may be assayed in every automated method is dependent on the speed of the tablet dissolution. Several mixtures were prepared during our experimental studies and a 50% methanol-water solution made slightly acidic was found to be the best in all respects. During the solubility studies, it was observed that the UV spectrum of a one-day-old alcoholic solution of hydralazine
Table 11. Stability of Hydralazine Hydrochloride in 50 % Methanolic Solution Colorimetric assay, Ultraviolet assay, absorbance absorbance Time in at 530 nm at 250 nm mine 3 0.425 0.660 10 0,420 0.640 20 0.420 0.620 30 0.420 0.610 Time is measured from point of hydralazine HC1 dissolution in 50% methanol. 5
hydrochloride was different from that of the fresh solution. No change was observed for hydrochlorothiazide in alcoholic solutions. In the case of hydralazine hydrochloride, the change definitely shows an interaction with the solvent; however, the nature of this interaction is unknown. Fortunately, it has no influence on the colorimetric procedure (See Table 11).
The Fluorometric Reserpine Assay. The fluorometric determination of reserpine alone and in tablet formulations is described in previous publications (3, 4) using both manual and automated methods. Tests were performed in our ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
567
~~
Table ILL Effect of Vanadium Pentoxide on Reserpine Assay Containing Hydralazine Hydrochloride in Alcohol Z Fluorescence ml satd Reserpine vzos Reserpinea hydralazinea
+
48 3 0
1 4
8
3 11 35 33
15 a
10-ml solution containing 1 pg/ml reserpine, 10-ml solution containing 1 pg/ml reserpine and 260 pg/ml
hydralazine HCl.
Table IV. Effect of Hydralazine Hydrochloride on Reserpine Assay Hydralazine Reserpine % Hydralazine error in mg/100 ml F.S. ff uores. concn. reserpine 5.0 20.5 80% low + 1 .o 12.5 20.8 50% low $2.5 22.5 20.4 10% low $0.5 25.0 20.3 +O.O% AO.0 27.5 20.2 10% high -0.5 -8.8 37.5 18.7 50% high 49.0
16.1
96% high
-26.2
laboratories to check whether the other two components, hydralazine hydrochloride or hydrochlorothiazide, have any effect on fluorescence and interfere with the reserpine assay. No appreciable fluorescence due to these compounds was observed. However, hydralazine hydrochloride reduces the vanadium pentoxide reagent, therefore requiring modification of the existing reserpine assay method. An unsuccessful attempt was made to separate hydralazine hydrochloride from reserpine by passing the solution through a cation exchange column. Apparently, the protonated column bound the basic reserpine onto its surface resulting in no fluorescence after the addition of vanadium pentoxide reagent. An alternative solution based on destroying the reducing ability of hydralazine hydrochloride before the reserpine determination was needed. The use of excess vanadium pentoxide reagent to oxidize the hydralazine hydrochloride seemed to be the simplest solution for this problem. From Table 111, it is evident that this solution is impractical. Since the vanadium pentoxide reagent is a saturated solution, the concentration of the reagent can be increased only by increasing its volume. This volume increase (about 8 times) lowers the sensitivity of the determination not only because of dilution but also because of a quenching effect. An automated version of this method was tried but the precision was poor. For this reason, a better method for the elimination of the hydralazine interference was desired. Complexing (19, 20) and oxidation (21) agents were investigated but with no success. i The most promising results were obtained by oxidation of hydralazine hydrochloride with hydrogen peroxide prior to the introduction of vanadium pentoxide. It was observed that the oxidation of hydralazine hydrochloride was rapid and colorless in the presence of hydrogen peroxide. Experiments proved that there is also an interaction between the unreacted hydrogen peroxide and the vanadium pentoxide. The ratio of hydrogen per(19) S. Fallab and H. Erlenmeyer, Heh. Chim. Acta, 40, 363 (1957). (20) S. Fallab, ibid.,45, 1957 (1962). (21) F. Fiegl, “Spot Tests in Organic Analysis,” Elsevier, Amsterdam, 1966, pp 271-2. 568
*
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
oxide to vanadium pentoxide was adjusted on the AutoAnalyzer to obtain maximum fluorescence, thereby assuring the presence of enough vanadium pentoxide to complete the reserpine oxidation. Unfortunately, even with the best reaction conditions, the fluorescence produced is dependent on the hydralazine concentration. The effect of a variation of hydralazine hydrochloride concentration on the reserpine results can be seen in Table IV. The proposed method is acceptable if the hydralazine hydrochloride content of the tablets does not exceed a +IO% range. Under this condition, the error found in the reserpine assay was not greater The reserpine assay is not affected by the than + O S % . hydrochlorothiazide content so that no special precaution regarding this component was necessary. The Colorimetric Hydralazine Assay. The most commonly used method for hydralazine hydrochloride determination is the iron complexing method (20, 22). This method was found unsuitable for automation because of disadvantages such as a large reagent blank due to the colored ferric ion, slow reaction rate, and the large number of reagents. The need for a new analytical method that was simpler and more precise than existing procedures became obvious. Since the complexing and reducing properties of hydrazine compounds are well known in the literature (23), it was decided to develop a new method which would be based on its reducing character. It was observed that a basic solution of hydralazine hydrochloride gave a highly colored solution with blue tetrazolium chloride. The color and its strength depend upon the solvent system used for the assay. The most permanent color was obtained when 50% ethanolic solution was used for hydralazine hydrochloride dissolution and the reagents were dissolved in USP ethanol. The variation of blue tetrazolium chloride concentration significantly influences the intensity of the absorption and the time of color development. Ethanol is the preferred solvent for this reagent since much higher concentrations were required for full color development when using absolute methanol. The order of reagent addition has little effect on the hydralazine absorption. The flow rates for the hydralazine hydrochloride section of the automated manifold were chosen so that the final analytical mixture was essentially in USP alcohol. As a result, the advantages of this solvent were retained in the automated version. A single dilution stage containing a 60-turn mixing coil assures complete mixing and allows time for full color development. Although the hydralazine hydrochloride determination is not affected by the reserpine and hydrochlorothiazide present in the formulation (see Table V), a slight increase in the result due to the placebo (tablet excipient material) occurred. This slight placebo effect can be eliminated by either subtracting it from the result or by adding it to the standard for very precise assays. This colorimetric assay obeyed Beer’s law when a standard containing the excipients and the other components was carried through the automated procedure. The UV Hydrochlorothiazide Assay. A UV method for the determination of a compound is generally applicable only in the absence of interferences due to the other compounds. While the absorption due to reserpine in this assay was negligible because of its low concentration, high interferences from hydralazine hydrochloride occurred because of overlapping absorption bands. Since the simplest and most common quantitative determination of hydrochlorothiazide ~
~
(22) P. Cooper, Pharm. J . , 177, 495 (1956). (23) F. C. Whitmore, “Organic Chemistry,” Second ed., Vol. 2, Dover Publications, New York,N.Y., 1951, p 660.
Hydralazine std. abs. 0.410 0.410 0.408 0.408 0.412 0.413 0.419 0.411 0.414 0.416 Av: 0.412 Range: 2 . 4 2
Table V. Effect of Reserpine and Hydrochlorothiazide on Hydralazine Assay Hydralazine Hydrochlorothiazide Reserpine concn. absorbance concn. 70% low 0.405 70% low 40% low 0.412 40% low 10% low 0.410 10% low Std 0.410 Std 10% high 0.407 10% high 50% high 0.409 50% high Av: 0.409 Range: 1.7%
Hydralazine absorbance 0.406 0.412 0.407 0.405 0.412 0.403 Av: 0.408 Range: 2.2%
Table VI. Effect of Reserpine and Hydralazine Hydrochloride on Hydrochlorothiazide Assay Hydrochlorothiazide Reserpine Hydrochlorothiazide Hydralazine Hydrochlorothiazide std. absorb. concn. absorb. concn. absorb. 0.470 0.462 70% low 0.456 70% low 0.468 0.464 40% low 0.463 40% low 0.465 0.463 10% low 0.467 10% low 0.464 0.470 Std 0.465 Std 0.464 0.464 10% high 0.465 10% high 0.463 0.465 50% high 0.470 50% high 0.463 0.465 Av: 0.464 Av: 0.464 0.464 Range: 3% Range: 1.1% 0.466 Av: 0.465 Range: 1.7%
Table VII. Comparison of Methanol with Ethanol on Reserpine, Hydralazine, and Hydrochlorothiazide Assay Reserpine Hydral- Hydrochlorofluoresazine thiazide Solvent cence, % absorbance absorbance 0.600 50% Ethanol 14.5 0.390 5 0 z Methanol 15.0 0.430 0.620
is based on its UV absorption (17), means were sought for its adoption. The different chemical properties of hydralazine hydrochloride and hydrochlorothiazide suggest a possible chromatographic separation. Cation exchange resins were investigated t o see if the cationic hydralazine would be bound onto the column and allow the slightly acidic hydrochlorothiazide t o pass through unchanged. This attempt was completely successful. As we know from the reserpine assay, this compound was also bound onto this column. The authors were confronted with the problem of how t o adopt this separation for the automated system. Several factors such as type of resin, particle size, length and diameter of the column must be considered. A macroreticular resin was chosen for its high efficiency, high porosity, and large surface area. These factors permit the use of short, high capacity columns that have a minimum resistance t o flow. The resin was sized t o 40-60 mesh for uniformity of packing into a narrow diameter tubing. Adjustment of the length and diameter of the column was critical as these factors affect both the absorbance and the resolution between samples. An ion exchange trap 15 cm in length and of 1 mm i.d. was found to be the best for all requirements. The column capacity was effective for about 150-200 assays and can be replaced with a fresh column within a few seconds.
Table VIII. Tablet Content Uniformity of Regular Ser-Ap-Es Production Batch Mg per tablet Number Serpasil Apresoline Esidrix 15.0 25.4 1 0.104 15.1 25.7 2 0.105 15.0 25.7 3 0.104 14.9 25.3 4 0.100 14.8 25.8 5 0.105 14.6 25.7 6 0.103 14.7 25.8 7 0.103 14.6 26.3 8 0.107 25.3 14.9 9 0.104 25.3 14.4 10 0.101 14.7 26.5 11 0.105 14.4 12 0.101 25.4 14.4 26.0 13 0.104 14.8 25.4 14 0.102 14.7 25.9 15 0.101 14.3 25.9 16 0.102 25.9 15.2 17 0.103 14.8 25.8 18 0.102 14.8 Mean : 0.103 25.8 4~1.7% Std dev: 4~1.7% 2~1.4% 14.6 Composite 0.102 25.3
Using the proper ion exchange column, the hydrochlorothiazide UV assay was completely unaffected by reserpine and hydralazine hydrochloride when their concentrations were varied over a wide range. These data are presented in Table VI. From this table, it is evident that a good separation was obtained using the small exchange column. The hydrochlorothiazide standard follows Beer’s law over a very wide range (2 mg t o 30 mg) in the presence of reserpine and hydralazine hydrochloride when using the automated method. ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
569
Time, second 60 68
120 180 240 540
Table E. Steady State Obtained for Three Components by Automated Assays Reserpine Hydralazine HC1 Hydrochlorothiazide % Steady Steady Steady % Fluores. state Absorbance state Absorbance state 19.2 20.3 21.5 21.5 21.5 21.5
89 94 100 100 100 100
0.430 0.448 0.464 0.475 0.477 0.477
In this assay a very simple mixing device was utilized, as suggested by Ayerst Laboratories, instead of the regular mixing coil. This gadget gives more efficient mixing, and can be used with or without air segmentation. Mixing is produced by the turbulence that results from sudden changes of velocity over a length of the analytical stream. Table VI1 shows a brief comparison using ethanol and methanol as solvents in the reserpine, hydralazine hydrochloride, and hydrochlorothiazide assays. As one can see from this table, the slight changes in fluorescence and absorption are insignificant from the analytical viewpoint and possibly can be explained by differences in flow rates between the two solvents. Several regular production batches, using 40 to 60 tablets from each batch, were analyzed by the fully automated sys-
90 94 97 99 100 100
0.490 0.512 0.590 0.610 0.620 0.620
79 83 95 99 100 100
tem. The results obtained by automated and compository assays are tabulated in Table VIII. The differences observed by the two methods are within experimental error and are in good agreement. Typical automation curves for reserpine, hydralazine hydrochloride, and hydrochlorothiazide were obtained. The symmetrical shape of these curves and the steady base line indicates the reliability of the automated method. Although sensitivity, except for the reserpine determination, was not the primary objective of this automation, the spectrophotometric measurements obtained are nearly of steady state intensity. The data obtained on steady state standards are summarized in Table IX.
RECEIVED for review August 13, 1971. Accepted October 5 , 1971.
Investigation of the Molecular Behavior of the Carr-Price Reaction Paul E. Blatzl and Andres Estrada Department of Chemistry, University of Wyoming, Laramie, W y o . 82070 and Pan American Coflege, Edinburg, Texas 78539 The blue color produced by the reaction of antimony trichloride with vitamin A and related compounds is the basis used for identification and determination of these compounds. This test is based on the work of Carr and Price. The reaction that takes place is complex and attempts to explain it have not been successful. In this investigation, retinol and retinyl acetate were allowed to react with antimony trichloride reagent at various temperatures and concentrations, and these reactions were followed spectroscopically. The resulting colored species were found to be the retinylic and anhydroretinylic cations identical to those reported in the literature for the reaction of polyenes with acids. Identification of these cations is used as a basis to suggest a pathway for the CarrPrice reaction.
IN 1926 CARRAND PRICEproposed a test for vitamin A based on the blue color obtained when antimony trichloride and vitamin A are mixed in chloroform as a solvent (1). The proposed test was based on a previous test developed by Rosenheim and Drummond (2), who had used sulfuric acid to obtain the blue color. The results with sulfuric acid were Present address, Department of Chemistry, The University of Missouri-Kansas City, Kansas City, Mo. 64110 (1) F. H. Carr and E. A. Price, Biochem. J . , 20,497 (1926). (2) 0. Rosenheim and J. C . Drummond, ibid., 19,753 (1925). 570
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
qualitative only. Carr and Price were able t o obtain quantitative results. This test eventually became the standard test for identification and quantitative determination of retinol (3), retinal (4), retinoic acid (5), 3-dehydroretinol (6),and other related compounds (3, 7). The brilliant blue color developed by these compounds and the chloroform solutions of SbC13is stable for approximately three minutes. Attempts t o stabilize the color for longer periods were unsuccessful ( I ) . Other studies have been carried out involving the reaction of certain acids with polyene compounds. Den0 et a f . (8, 9 ) and Sorensen (IO, 11) studied a series of aliphalic (3) J. G. Baxter, Progr. Chem. Org. Nutur. Prod., 9, 41 (1952). (4) and G. Wald, J . Gen. Physiol., ~, R. Hubbard. R. I. Gregerman, 36,415 (1953): (5) G . Katsui. S. Ishikawa. and M. Shimizu, J. Vitaminol. (Kyoto), 12,122 (1966). ( 6 ) H. H. Inhoffen, F. Bohlman, and M. Bohlman, Ann. Chem., 565, 35 (1949). (7) B. Conner Johnson, “Method of Vitamin Determination,” Burgess Publishing Co., New York, N.Y., 1949. (8) N. C. Deno, H. G. Richey, Jr., N. Friedman, J. D. Hodge, J. J. Houser, and C. U. Pittrnan, Jr., J . Amer. Chem. Soc., 85, 2991 (1963). (9) N. C. Deno, C. U. Pittman, Jr., and J. 0. Turner, ibid., 87, 2153 (1965). (10) T. S. Sorensen, Can. J . Chem., 42, 2768 (1964). (11) T. S . Sorensen, J . Amer. Chem. Soc., 87, 5075 (1965). \
,
