(16) A. H. Antone and D. F. Sayre, J. Pharmacol. Exp. Ther., 138, 360 (1966). (17) S. Udenfriend, D. F. Bogdanski, and D. F. Weissbach, Science, 122, 972 (1955). (18) P. T. Kissinger. C. Refshauge,R. Dreiling, and R. N. Adams, Anal. Left.,8, 465 (1973). (19) P. Albrecht, M. B. Visscher,J. J. Bittner, and F. Halberg, Proc. SOC.Exp. Biol. Med., 92, 702 (1956). (20) R. J. Borgman, M. R. Baylor, J. J. McPhillips,and R. E. Stitzel, J. Med. Chem, 17, 427 (1974). (21) Louis Levine, "Biology of the Gene", 2d ed., C. V. Mosby Co., St. Louis, Mo., 1973. (22) R. M. Flemina, W . G. Clark, G. D. Fenster, and J. E . Towne, Anal. Chem., 37, 292 (1963). (23) R. G. Wiegand and J. E. Perry, Biochem. Pharmacol., 7, 181 (1961). (24) C. B.Smith, J. Pharmacol. Exp. Ther., 142, 343 (1963). (25) H. C. Agrawai, S.N. Glisson, and W. A. Himwich, lnt. J. Neuropharmacol., 7, 97 (1968).
(26) S. T. Weintraub, W. B. Stavinoha, R. L. Pike, W. W. Morgan, A. T. Modak, S. H.Koslow, and L. Blank, Life Sci., 17, 1423 (1975). (27) R. D. Ciavanelio, R. E. Barchas, G. S. Byers, D. W. Stemmle, and J. D. Barchas, Nature (London),221, 363 (1969). (28) C. Refshauge, Ph.D. Thesis, University of Kansas, Lawrence, Kan., 1974. (29) H. Alliger, Am. Lab., No. 7, 75 (1975). (30) R. H.Cox and J. L. Perhach, Jr., J. Neurochem., 20, 1777 (1973). (31) R. P. Maickel in ':Methods of Neurochemistry", Voi. 2, R. Fried, Ed,, Marcel Dekker, New York, 1972. (32) C. L. Blank, J. Chromatogr., 117, 35 (1976).
RECEIVEDfor review September 7,1976. Accepted December lg7" This work was by grant number MH26866-01, awarded by NIMH/DHEW. 8 j
Separation of Five Major Alkaloids in Gum Opium and Quantitation of Morphine, Codeine, and Thebaine by lsocratic Reverse Phase High Performance Liquid Chromatography C. Y. WU" and J. J. Wittick Merck Chemical Manufacturing Division, Rahway, N.J. 07065
Two similar isocratic liquld chromatographic (LC) systems have been developed for the determination of morphine, codeine, and thebaine in gum opium. One Is for the quantitatlve determination of morphine alone and the other for the simultaneous quantltative determination of codelne and thebaine. Two 30-cm X 4.0-mm i.d., p-Bondapak C18/Porasll columns are used for both systems which differ only In the composition of the mobile phase used. The composltlonof the mobile phase used for the morphine determination is 0.1 M NaH2P04in 5 % CH3CN/H20 and for the codelne and thebaine determination Is 0.1 M NaH2P04In 25% CH3CN/H20. Both systems are very reproduclble and sultable for routlne anaiysls. The preclslon of the method for morphine is 0.8% relative standard deviation and for codelne and thebaine 1.3 and 3.3 % relatlve standard deviation, respectively.
In spite of the fact that opium has been associated with human activities for centuries, chemists are still searching for better analytical methods to measure the alkaloids content of this important pharmaceutical raw material. Progress was hampered primarily because there are more than twenty alkaloids present in gum opium and suitable techniques for separating this complex mixture were lacking. However, with the development of a variety of chromatographic techniques, these difficulties have been overcome. Tedious prior purification of a specific alkaloid before quantitation is no longer necessary and simultaneous quantitative analysis for several alkaloids in the complex mixture has become a reality. Steady progress in the use of various chromatographic techniques for the analysis of opium and related mixtures has been demonstrated in the literature (1-18). In 1973 Wu et al. (7), using a synthetic mixture of five of the major opium alkaloids, successfully demonstrated that the HPLC technique is potentially useful for such analysis. In the same year, Wittwer (16) reported a gradient HPLC system which he claimed to be suitable for the determination of opium alkaloids. However, only limited data were reported. Later, Beaseley et
al. (15)described a procedure using normal phase liquid-solid adsorption chromatography with gradient elution. The method is lengthy and, unlike reverse phase chromatography, separation by normal phase liquid-solid adsorption chromatography is highly susceptible to the water content both of the adsorbent and of the mobile phase (19), making the technique difficult to use for routine analysis in our opinion. Techniques for opium alkaloids analysis other than chromatography are also available. For details refer to the literature (20-25). The objective of this paper is to introduce a practical, specific, relatively simple and rapid method with the capability of yielding precise quantitation for morphine, codeine, and thebaine in gum opium. Two essentially identical isocratic liquid chromatographic systems are described, one for morphine and the other for codeine and thebaine. The chief difference in these two systems is in the composition of the mobile phase. A microparticulate, w-Bondapak C l g column was selected and used because it provided the high column efficiency which is necessary for the analysis of a coplex mixture such as gum opium. This column also provided excellent reproducibility over a long period of time, making it especially suitable for routine analysis.
EXPERIMENTAL Apparatus. A DuPont Liquid Chromatograph Model 830, a Schoeffel SF 770 Spectroflow Monitor equipped with a deuterium lamp, an Autolab Vidar 6300 Digital Integrator, and a HewlettPackard strip chart recorder Model 7123A were properly connected and used. Samples were introduced through an injection valve purchased from DuPont Instruments (Wilmington, Del., Catalogue No. 204590). Reagents a n d Solutions. Pure morphine sulfate pentahydrate, pure codeine phosphate, and pure thebaine standards were obtained within the Merck Chemical Manufacturing Division. Reagent grade calcium hydroxide powder, 85%phosphoric acid, glacial acetic acid, sodium chloride, sodium perchlorate, dimethyl sulfoxide, and methylene chloride were used. Acetonitrile (UV) "Distilled in Glass" was purchased from Burdick & Jackson, (Muskegon, Mich.). Presaturated calcium hydroxide solution was prepared fresh daily by ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
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f2: I
b
IO TIME
PO (MINUTE)
30
I
Figure 1. Separation of five of the major opium alkaloids with pH 4.8 mobile phase Solution: A mixture of five standard alkaloids in 10 ml mobile phase (morphine sulfate 11.4 mg, codeine alkaloid 5.5 mg, thebaine alkaloid 7.2 mg, papaverine hydrochloride 5.0 mg, noscapine alkaloid 10.0 mg). Mobile phase: 0.1 M NaH2P04in 25% CH3CN/H20pH 4.8. Detector: 254 nm UV (DuPont). Flow rate: 1.25 ml/min. Pressure: 2000 psig. Peak identity: (1) Morphine, (2) codeine, (3) thebaine, (4) papaverine, apd (5) noscapine
mixing a saturated aqueous solution of calcium hydroxide with methylene chloride. Presaturated methylene chloride was prepared by mixing small but excess quantities of calcium hydroxide and water with methylene chloride. Mobile Phase Preparation. The mobile phases used for morphine (0.1 M NaH2P04 in 5% CH3CNIH20) and codeidthebaine (0.1 M NaHZP04 in 25% CH&N/H20) were prepared by mixing appropriate quantities of acetonitrile and prefiltered 0.4 M NaH2P04 and diluting to volume with prefiltered distilled water. Filtration to remove particulate matter was achieved by passing the solutions through MF Millipore filters with a pore size of 0.45 Fm (HAWP 04700). Chromatographic Conditions. Column: F-Bondapak Clg/Porasil, two 30-cm X 4.0-mm, i.d., (Waters Associates); Mobile phases-as above, Flow rate: 1.25 ml/min (2000 psig) for morphine and 1.45 ml/min (2500 psig) for codeine and thebaine; Detector: 254 nm (DuPont) or 286 nm (Schoeffel); Injection volume: 10 p1 valve injection. Any differences from the described conditions are noted in the caption of each chromatogram. Sample Preparation. Two grams of gum opium test sample, accurately weighed, was allowed to soak overnight in 20 ml of water. The resulting slurry was completely dispersed by stirring and by ultrasonic agitation. The supernatant was separated by centrifugation and transferred to a labeled 50-ml volumetric flask 1. The solids were thoroughly washed two more times using 15- and 10-ml portions of water and, after centrifugation, the supernatants were added to flask 1. The solution in flask 1 was diluted to volume with water before being used in subsequent analyses. Morphine Determination. A 20.0-ml aliquot from flask 1 was extracted in the presence of 0.2 g calcium hydroxide powder with 10.0 ml of methylene chloride using high speed centrifugation to separate the phases. (The calcium hydroxide is used to remove water-soluble resinous matter by forming an insoluble cake and also serves to convert morphine to the phenolate anion whereby it is retained in the aqueous phase.) The extract was cooled to room temperature and a 15-ml aliquot of the aqueous phase promptly pipetted into a 25-ml volumetric flask containing 10 drops of glacial acetic acid and carefully diluted to volume with water. Any turbidity occurring during the dilution was cleared by the addition of a few more drops of glacial acetic acid before diluting to volume. The solution was filtered through a Millipore filter (HAWP 01300, pore size 0.45 pm) and analyzed chromatographically. 360
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
10
0
40
TIME
20
(MINUTE)
Figure 2. Separation of five of the major opium alkaloids with pH 2.0 mobile phase Mobile phase: 0.1 M NaH2P04in 25 YO CH3CN/H20 pH adjusted to 2.0 with 85 'YO H3P04.Peak identity: (1)Morphine, (2) codeine, (3) thebaine, and (4) papaverine -t noscapine. For solution, detector, flow rate, and pressure, see Figure 1
I , 0 IO PO 30 TIME
40
$0
L
00
(MINUTE)
Flgure 3. Separation of five of the major opium alkaloids with pH 7.10 mobile phase Mobile phase: 0.1 M NaH2P04 In 25% CH&N/H20 pH adjusted to 7.10 with 10% NaOH. Peak identity: (1) Morphine, (2) codeine, and (3)thebaine. Note: papaverine and noscapine were not eluted within 1 h. For solution, detector, flow rate, and pressure, see Figure 1
Codeine and Thebaine Determinations. A 25-ml aliquot of the aqueous opium extract from flask 1was extracted in the presence of 0.2 g of calcium hydroxide powder and 5 g of sodium chloride with 10.0 ml of methylene chloride. After phase separation using high speed centrifugation, a 4.0-ml aliquot of the methylene chloride phase was diluted to 25 ml with presaturated methylene chloride and mixed. The entire methylene chloride solution was backwashed with 20 ml of presaturated calcium hydroxide solution and filtered through a Millipore Mitex filter (LSWP 01300, pore size 5 pm) using a syringe fitted with a Swinney adapter. Exactly 15.0 ml of the filtrate was evaporated to dryness and then redissolved in 10.0 ml of the mobile phase to be used with the aid of a few drops of 85% phosphoric acid to ensure complete solution. This solution was filtered through a Millipore Fluoropore filter (FHWP 01300, pore size 0.45 pm) and analyzed chromatographically. (This solution may slowly develop a pink color probably due to the presence of some unstable phenolic alkaloids or pigments which are extracted along with the codeine and thebaine. The color change does not affect the codeine and thebaine assays.) Standard Preparation. Morphine. Morphine sulfate pentahydrate containing the equivalent of 120 mg morphine alkaloid was weighed accurately and dissolved in 25.0 ml of water. A 20.0-ml aliquot was extracted in the presence of 0.2 g calcium hydroxide with 10 ml
Table I. Effect of pH of Mobile Phase on Retention of Alkaloids
Table 11. Exhaustive Extraction of Opium Alkaloids from a Gum Opium Sample
Retention time, min pH of mobile phase
Morphine
2.0 4.8 7.1
5.0 5.4 6.0
Codeine
Thebaine
6.0 6.6 9.6
12.3 15.5 33.6
Papaverine 18.5 33.0 >60
Noscapine 18.5 38.0 >60
methylene chloride. This step is t o simulate the sample preparation so as to compensate for any volume change. After phase separation by high speed centrifugation, a 15-ml aliquot of the aqueous phase was pipetted into a 25-ml volumetric flask containing 10 drops of glacial acetic acid and carefully diluted to volume with water. About 10.0 ml of this solution was filtered through a MF Millipore filter (HAWP 01300, pore size 0.45 gm) for use as the morphine standard. Codeine and Thebaine. Codeine and thebaine standards containing the equivalent of 2 mg codeine alkaloid and 12 mg thebaine alkaloid were accurately weighed into a 25-ml volumetric flask. After the addition of about 10 ml of mobile phase, 10 drops of 85%phosphoric acid was added to facilitate dissolution of the alkaloids and the solution was diluted to 25.0 ml with the mobile phase being used. Quantitation. Quantitation was accomplished by comparison of the area under the desired sample peak with that of a standard. Valve injections were used for all the quantitative work. Column Regeneration. It is recommended that the column be regenerated after about every 50 morphine determinations. If the column is used only for thebaine and codeine, less frequent regeneration will be required because the latter sample solutions are relatively free of excipients. The regeneration can be achieved by flushing the column with approximately 150 ml of mobile phase consisting of 0.1 M NaH2P04 t 0.05 M NaC104 in 25% CHzCN/H*O,pH 3.0 (adjusted with H3POJ. The column can be regenerated repeatedly until it fails to perform, as indicated by inadequate separations.
R E S U L T S A N D DISCUSSION ' Ideally, we would like to have a simple and reproducible liquid chromatographic (LC) method capable of determining all of the five major opium alkaloids in gum opium in a single chromatographic run and requiring only minimal sample preparation. After intensive investigation, we were unable to find a simple approach which would accomplish this. With simplicity and reproducibility still in mind, we discarded the ideas of using (a) an ion-exchange LC approach because we found that ion-exchange column packing material, especially anion-exchange types, exhibited notoriously poor reproducibility, (b) a gradient elution LC technique because this technique is not yet a recommended tool for precise quantitation and, even if it were precise, it is not a convenient technique for routine analysis, (c)" the idea of employing a mixed-organic solvent, such as, 15% isopropyl alcohol in chloroform, for multiple extractions of all the alkaloids before chromatography because this approach is tedious and the precision of the morphine determination may be impaired due to excessive sample manipulation. In view of these drawbacks, we developed two similar isocratic LC methods, one for morphine and the other for codeine and thebaine, using a durable p-Bondapak CIS column. The two LC systems differ only in the composition of the mobile phase and are suitable for handling relatively crude sample solutions. Most important of all, the methods are relatively rapid and highly reproducible. It should be noted that the method for codeine and thebaine also appears to be suitable for noscapine and papaverine (Table I). However, no quantitative data for these two alkaloids are presented in the paper. Chromatography. Using two 30-cm X 4.0-mm, i.d. p-
Sequence of extraction
Volume of water % of Total alkaloid content used for each removed from gum opium extraction, ml Morphine Codeine Thebaine
1 2
20 15
3 4 5
10 10 10
90.6 7.70 1.52 0.18 0
87.2 10.6 2.20 0 0
85.9 8.21 4.30 1.29
0.27
Table 111. Extraction Efficiency of Methylene Chloride for Codeine and Thebaine from Aqueous Opium Extract (1 g Opium in 25 ml) Procedure Single extraction (1 X 10 ml) Double extraction (2 X 10 ml)
Alkaloids found, % Codeine Thebaine 3.82
2.07
3.77
2.08
Bondapak C18 columns connected in series, and the mobile phase, 0.1 M NaH2P04 in 25% CH$N/HzO, baseline separation of five of the major opium alkaloids is achieved (Figure 1). However, morphine is eluted very near the solvent front, making this system unsuitable for morphine determination. By lowering the percentage of CH3CN in the mobile phase to 5%, the system is, nevertheless, readily adaptable to morphine determinations. With this last mentioned mobile phase, morphine is retained on the column long enough to permit its separation from other interferences. The other alkaloids are strongly retained under these conditions and are eluted a t some later time. In practice, the major alkaloids other than morphine are removed from the sample solution by a simple liquid-liquid extraction to facilitate a rapid analysis for morphine. Although the retention and separation of the major alkaloids on the CIScolumn are primarily governed by the amount of acetonitrile in the mobile phase, they are also affected by the pH of the mobile phase. Figures 1 through 3 demonstrate the effect of the p H of the mobile phase on the retention and the separation of the alkaloids. A flow rate of 1.25 ml/min was used for this study. Figure 1 shows that baseline separation of papaverine and noscapine was achieved with a mobile phase of p H 4.8 (0.1 M NaHZP04 in 25% CHsCN/HzO). Figure 2 shows that no such separation was possible with a pH 2.0 mobile phase (adjusted with 85% H3PO4) where the retention of each alkaloid is shorter, compared with that obtained with the pH 4.8 mobile phase. Figure 3 shows that with a pH 7.10 mobile phase (adjusted with 10% NaOH), papaverine and noscapine were retained on the column even after one hour and that longer retentions are observed for the other three alkaloids as well. Although the chromatogram was not run long enough to allow noscapine and papaverine to elute off the column, on the basis of the separation pattern of three components that were eluted, it would seem reasonable to predict that noscapine and papaverine would be well separated with the pH 7.10 mobile phase. In short, the opium alkaloids appear to be more strongly retained with a high p H mobile phase than with a low pH mobile phase on a CIS column. Table I summarizes these results. Analysis. Morphine, codeine, and thebaine can be effectively extracted from opium with water at room temperature. ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
361
'i I\I\\
I N S T R U M E N T : GARY 118 THEBAINE 0.100mrnolar
A
O ' T 0.8
-
0.7
0.1 I U
: z a : 0,e-
0.1
0
MORPHINE 0.2e4rnmolor
ul
e m
0.4-
0.3
IN 0.IM NaH,?O,
-
0.t-
0. I
-
20
IO
0
SOLVENT: MORPHINE: 6 U O H r G N In 0 . I Y NmH,W4
TIME ( M I N U T E I
2 1 % OH3CN IN 0 . I M NnH,PO, 6 % OH, CN IN 0.1 M NO N, PO,
v. H t O
240
280
'
0.0-
260
Figure 4. Typical chromatogram of aqueous opium extract Sample: Indian gum opium containing the equivalent of 0.6 g opium in 25 ml final solution. Detector: UV 286 nm (Schoeffel SF 770). Chromatographic conditions: see text. Peak identity: (1) Morphine
ZeO
270
WAVELENGTH
Figure 6. baine
UV
290
3M)
310
320
W
340
(nm)
absorption spectra for morphine, codeine, and the-
Table IV. Reproducibility of LC Method
f
f:
I
Day
Morphine
Codeine
Thebaine
1 2 3 4 5 6 7
10.67
3.97 3.95 3.84 3.94 3.93 3.87 3.87 3.91 0.05 1.3%
1.99 1.93 2.02 1.92 2.07 2.03 2.09
Av
Std dev Re1 std dev
3
I
0
I
10
20 TIME
30
40
SO
60
(MINUTEI
Figure 5. Typical chromatogram of methylene chloride opium extract after being evaporated and redissolved in the mobile phase Sample: Indian gum opium containing the equivalent of 0.24 g opium in 10 ml final solution. Detector: UV 286 nm (Schoeffel SF 770). Chromatographic conditions: see text. Peak identity: (1) Codeine, (2)thebaine, (3)papaverine and (4) noscapine
The extraction efficiency of five successive extractions with water is presented in Table 11.Each aliquot of aqueous extract was treated as a separate sample and the alkaloids content was then determined by following the procedure described in the Sample Preparation section. The data (Table 11) show that adequate quantitative extraction of the three alkaloids from 2 g of gum opium is attained after three successive extractions with 20,15, and 10 ml of water. Codeine and thebaine are readily extracted from basic aqueous media into methylene chloride. I t was found that a volume of 10 ml of methylene chloride is adequate for the quantitative removal of codeine and thebaine from a 25-ml aqueous extract of 1 g of gum opium (Table 111);multiple 362
ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
10.45
10.50 10.60 10.67 10.62 10.52 10.59 0.08 0.76%
2.01
0.066 3.3%
extractions with methylene chloride are obviously unnecessary. To substantiate the effectiveness of the aqueous extraction, the opium residue left after the three successive extractions was analyzed directly by HPLC. The residue was completely dissolved in 5 ml of dimethyl sulfoxide (DMSO) by heating for 20 min on a steam bath. The solution was then diluted to 10 ml with DMSO. A 4-ml aliquot was diluted to 25 ml with water, filtered, and subjected to HPLC analysis for morphine. Results indicated that 0.5% of the total amount of morphine in the opium remains in the residue as compared with 0.2% calculated from the data ip Table I. In addition, a known amount of added morphine was recovered with 100% efficiency from the DMSO solution of the residue. Thus, the method provides a t least 99.5% recovery of morphine from opium. In a similar manner, the DMSO solution of residue, diluted with water and filtered, was extracted with methylene chloride to remove any remaining codeine and thebaine. The methylene chloride was evaporated to dryness, redissolved in mobile phase, and examined by HPLC. Appropriate standards were carried in parallel through this procedure. Approximately 2% and 4% of codeine and thebaine, respectively, remain in the residue compared with 0% and 1.6% calculated from the data in Table I. Such recoveries are adequate considering the levels of these alkaloids present in opium but could be improved if desired by increasing the number andlor volume of the aqueous extractions. Figure 4 is a typical chromatogram of an aqueous opium
extract that was washed with methylene chloride under basic conditions to remove all the methylene chloride-extractable opium alkaloids from the aqueous phase, leaving morphine in the aqueous phase. Acidification of the aqueous sample solution before injection is necessary because the mobile phase is acidic (pH 4.8); injecting a basic sample solution into an acidic mobile phase may create chromatographic anomalies, such as band broadening, due to sample precipitation a t the column head. Figure 5 depicts a typical chromatogram of methylene chloride extract after the organic solvent was evaporated and the residue redissolved in the mobile phase. The reproducibility of the method is summarized in Table IV. The results were obtained by analyzing the same sample, prepared daily, for seven consecutive days. As can be seen, the two methods are quite precise for the determination of morphine and codeine. Somewhat lower precision was obtained for thebaine, since a relatively crude integrator was used for this work. Better precision for thebaine might be possible with. a more sophisticated data processor which could compensate, to some extent, for the poorer definition of the thebaine peak. Linear peak area response was obtained both at 254 and 286 nm for injection of up to at least 80 Fg of morphine, 25 pg of codeine, and 20 pg of thebaine. For the same quantities injected, only codeine gave linear peak height response. Precaution should be taken in this regard if peak height is to be used for quantitation. Although both 254 nm and 286 nm were used in this work, it is recommended that 286-nm detection be used whenever possible. The UV absorption spectra indicate that 286 nm is a common absorption maximum for the three major alkaloids in the mobile phase employed (Figure 6).
LITERATURE CITED (1)E. Stahl, Ed., "Thin-Layer Chromatography", Springer-Verlag, New York, N.Y., 1969,p 436. (2)D. Furmanec, J. Chromafogr., 89, 76 (1974). (3)A. Bechtel, Chromafographia, 5, 404 (1972). (4)K. D. Parker, C. F. Fontan, and P. L. Kirk, Anal. Chem., 35, 356 (1963). (5)S. Yamaguchi, I. Seki, S. Okuda, and K. Tsuda, Chem. Pharm. Bull., 10, 755 (1962). (6)H.A. Lloyd, H. M. Fales, P. F. Highet, W. J. A. VandenHeuval, and W. C. Wildman, J. Am. Chem. SOC.,82,3791 (1960). (7)C. Y. Wu, S.Slggia, T. Robinson, and R. D. Waskiewicz, Anal. Chim. Acta, 63,393 (1973). (8)J. H. Knox and J. Jurand, J. Chromatogr., 82,398 (1973). (9)J. H. Knox and J. Jurand, J. Chromatogr., 87,95 (1973). (10)P. J. Twitchett, J. Chromatogr., 104,205 (1975). (11) R . Verpoorte and A. B. Svendsen, J. Chromatogr., 100,227 (1974). (12)R. Verpoorte and A. B. Svendsen, J. Chromatogr., 100,231 (1974). (13)W. B. Caldweil, United Nations Secretariat, STISOAISER. J/17,(Nov.
. -. -,.
107A\
D. W. Smith, T. H.Beasley, Sr., R. L. Charles, and H. W. Ziegler, J. Pharm. Sci., 62, 1691 (1973). T. H. BeasleY, D. W. Smith, H. W. Ziegler, and R. L. Charles, J. Assoc. Off. Anal. Chem., 57,85 (1974). J. D. Wittwer, J. Forensic Sci., 18, 138 (1973). H. W. Ziegler, T. H. Beasley, Sr.. and D. W. Smith, J. Assoc. Off. Anal. Chem.. 57. 85 11975). (18)E. Murgia and H. F. Walton, J. Chromatogr., 104,417 (1975). (19)L. R. Snyder and J. J. Kirkland, "Introduction to Modern Liquid Chromatography", John Wiley and Sons, New York, N.Y., 1974,pp 239-281. (20)E. Brochmann-Hanssen, Medd. Norsk. Farm. Selsk., 17, 76 (1955). (21)E. Brochmann-Hanssen and A. B. Svendsen, J. Pharm. Sci., 52, 1134
(1963). (22)C. H.VanEtten, F. R. Earle, T. A. McGuire, and F. R. Senti, Anal. Chem., 28, 867 (1956). (23)United States Pharmacopeia, 19th Rev., Mack PublishingCo., Easton, Pa., 1975,p 350. (24)H. Bohme and R. Strohecker, Arch. Pharm., 285,422 (1952). (25)H. Bohme and R . Strohecker, Arzneim.-Forsch., 3, 236 (1953).
RECEIVEDfor review July 28,1976. Accepted November 24, 1976.
Quantitative Determination of StabiIizers in Tetraethylene Glycol Dimethacrylate by High Pressure Liquid Chromatography G. A. Pasteur Bell Laboratories, 600 Mountain Avenue, Murray Hill, N.J. 07974
A quantitative determlnatlon of 4,4'-thiobis(3-methyl-6-terfbutyiphenol) (TMTPB), methyl ether of hydroqulnone, and hydroquinone in tetraethylene glycol dimethacrylate (TEGDMA) has been achieved uslng liquid chromatography on 10-p porous silica particles and UV detectlon. interference due to polymerizationof TEGDMA induced by UV radiation from the detector is avoided by deflecting the UV beam when the preceding refractive index detector signals the elution of the monomer from the column. The detection limits are 15,9, and 21 ng for TMTBP, MEHQ, and HQ, respectlvely. Repllcate analyses of commercially available formulations had a precision (average deviation) of 5 % for concentrations above 50 ppm and 10% for concentrationsbelow 25 ppm.
Tetraethylene glycol dimethacrylate (TEGDMA) is used as a cross-linking agent in some polymer formulations ( I , 2). Hydroquinone (HQ) and methyl ether of hydroquinone (MEHQ) are used as TEGDMA polymerization inhibitors ( 3 ) which must be present a t high enough levels to ensure good shelf life and thermal stability during polymer processing, but
low enough not to interfere significantly with the cross-linking 4,4'-Thiobis(3-methyl-6-tert-butylphenol) reaction. (TMTBP) (Santonox) is the usual antioxidant included in TEGDMA. Commercially available TEGDMA must be characterized analytically to ensure that these protective additives are present at effective levels. Gas chromatography is not suitable for the analysis. Experiments a t the temperature necessary for elution and separation showed that TMTBP degrades and its degradation products obscure the chromatogram. Liquid chromatography (an ambient temperature process) has been chosen for this determination. A reliable analytical procedure for the quantitative determination of HQ, MEHQ, and TMTBP is described in this report.
EXPERIMENTAL T h e solvent delivery system is a Waters Associates M o d e l 6000 pump. A 25-cm Si-10 M i c r o p a k column (Varian) (1.5-mm id., 3.0-mm 0.d.) is used for t h e separations. T w o types o f syringes are used: 5-pl Pressure-Lok B-110 a n d 5 - p l H a m i l t o n 700 in conjunction w i t h a septum injector. A Waters Associates R 401 differential refractometer is used in series w i t h a V a r i a n 635 U V - v i s i b l e spectrophotometer
ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
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