Table I I . Tropane Alkaloids Reten- Retention tion time, volume. min mil
Name
Scopolamine hydrobromide Eucatropine hydrochloride Apoatropine hydrochloride Homatropine sulfate Atropine sulfate Hyoscyamine hydrochloride (optically active form of atropine) Tropine
7.2 7.5 14.4
20 21
21.6 12
IJV sensitivity. integrator counts per p g
5.9 6.2 11.8 16.4 17.2
23.4 29.8 1320 18.7 45.5
17.7
16.5
9.9
alkaloids utilizing an industrially available high speed, high pressure liquid chromatograph. The work reported here shows the applicability and usefulness of high speed, high pressure liquid chromatography to the field of pharmaceutical analysis. It is especially valuable in the case where the sample would have to be converted to its free base before it could elute from a gas chromatography column, as would be true with the above alkaloid salts. Other advantages over gas chromatography would be the performance of the analysis a t room temperature and the ability to scale up the system, with relatively few modifications, in order to obtain preparative samples. With the utilization of high speed, high pressure liquid chromatography, the shortcomings of gas chromatography can be bypassed without loss of efficiency or time.
CONCLUSION The reported results show the separation and quantitation of a number of pharmacologically important similar
Received for review November 30, 1972. Accepted April 30, 1973.
Formation of Trimethylsilyl Derivatives of Tetracyclines for Separation and Quantitation by Gas-Liquid Chromatography Kiyoshi Tsuji and John H. Robertson Control Analytical Research and Development, The Upjohn Company, Kalamazoo, Mich. 49001 Following reports which implicated degradated tetracycline compounds, especially 4-epianhydrotetracycline, in renal dysfunction (1, 2 ) , a large number of chromatographic procedures concerning the separation and identification of tetracyclines has appeared. The analytical methods reported are based on thin layer chromatography (38), paper chromatography ( 9 ) , gel filtration ( I O ) , or partition column chromatography (11-15). Recently, an automated liquid chromatographic method has been reported (16). The method is an automation of the previously reported diatomaceous earth column method (12). In 1969 the British Pharmacopoeia (BP) (17) established a strict limit for 4-epitetracycline (ETC), anhydrotetracycline (ATC), and 4-epianhydrotetracycline (EATC)
J. M. Gross, Ann. Intern. Med., 58, 523 (1963) L. I. Ehrlich and H. S. Stein, Pediatrics, 31, 339 (1963). P. P. Ascione. J. B. Zagar, and G. P. Chrekian, J. Pharm. Sci., 56, 1393 (1967). I. C. Diykhuis and M. R. Brommet, J, Pharm. Sci., 59, 558 (1970). A. A. Fernandez. V. T. Noceda, and E. S. Carrera. J. Pharm. Sci., 58, 443 (1969). P. B. Lloyd and C. C. Cornford, J. Chromatogr., 53, 403 (1970). Y . Nishimoto, E. Tsuchida. and S. Toyoshima, J. Pharm. SOC.,Jap., 87, 516 (1967). D. L. Simmons, R. J. Ranz, H. S. L. Woo, and P. Picotte, J. Chromatogr., 43, 141 (1969). A. Sina, M . K. Youssef, A. A. Kassem. and I . A. Attia, J. Pharm. Sci., 60, 1544 (1971). B. W. Griffiths, R. Brunet, and L. Greenberg, Can. J. Pharm. Sci., 5 , 101 (1970). P. P. Ascione and G. P. Chrekian, J. Pharm. Sci., 59, 1480 (1970). P. P. Ascione. J. B. Zagar, and G. P. Chrekian J. Pharm. Sci., 56, 1396 (1967). W. W. Fike and N. W. Bake. J Pharm. S c : , 61. 615 ( 1 9 7 2 ) . R. G. Kelly, J. Pharm. Sci., 53, 1551 (1964). V . C. Walton, M. R. Howlett, and G. B. Selzer. J. Pharm. Sci., 59, 1160 (1970). P. P. Ascione, J. 8. Zagar. and G. P. Chrekian, J. Chromatogr., 65, 377 (1972). "British Pharmacopoeia 1968," Addendum, 1969, p 77.
2136
in tetracycline (TC) based principally on the procedure reported by Ascione et al. ( 3 ) . However, the detection limit of the method (5%) (3) and the procedure, according to Lloyd and Cornford ( 6 ) , is tedious, the results are variable, and, in particular, the limit of detection, according to them, appears to be optimistic. The detection limit of the diatomaceous earth column method is approximately 1% due primarily to the spectrophotometric read-out system in use (18). The proposed FDA limit for EATC is not more than 3% (19). Obviously, there is a need for a sensitive method which separates and precisely quantitates various tetracycline entities with speed, thereby making the method suitable for a laboratory where a large quantity of tetracycline preparations are analyzed routinely. Gas-liquid chromatography (GLC) has been applied as a sensitive method for the separation and quantitation of various antibiotics and their isomers and derivatives (2026). This report is, to the best of our knowledge, the first to describe trimethylsilylation of tetracyclines and their separation and quantitation by GLC.
EXPERIMENTAL Apparatus. An F & M M o d e l 400 gas c h r o m a t o g r a p h w i t h a f l a m e i o n i z a t i o n detector was used: gas flow rate, h y d r o g e n 40 ml/min, a i r 600 ml/min, a n d h e l i u m 55 ml/min; c h a r t speed, 0.25
(18) J. D. Hettinger, The Upjohn Company. Kalamazoo, Michigan, personal communication, 1968. (19) Fed. Regisf., 34, (141) 12286, (1969). (20) T. Endo and H. Yonehara, J. Antibiot., 23, 91 (1970). (21) C. Hihta, D . L. Mays. and M. Garofalo, Anal. Chem., 43, 1530 (1971). ( 2 2 ) R. L. Houtman, D . G. Kaiser, and A. J. Taraszka, J, Pharm. Sci., 57,693 (1968), (23) M. Margosis, J. Chromatogr., 47, 341 (1970). (24) K. Tsuji and J. H. Robertson, Anal. Chem., 41, 1332 (1969). (25) K. Tsuji and J. H. Robertson, Anal. Chem., 42, 1661 (1970). (26) K. Tsuji and J. H. Robertson, Anal. Chem., 43, 818 (1971).
A N A L Y T I C A L CHEMISTRY, VOL. 4 5 , NO. 1 2 , OCTOBER 1 9 7 3
100,
75
>
t v) z
W
+
714
5 50 W
P k-
I
565
a
709
-1 W r
25
560
660
550
640
m/e
Figure 1. Mass spectrum of TMS-TC
inch/min; oven temperature, 260 "C; flash heater, 260 "C; detector temperature, 290 "C. Column. A glass column 3 mm i.d. X 1850 mm (6 ft) packed with 3% JXR (methylsilicone) on Gas Chrom Q 100-120 mesh (Applied Science Laboratories, Inc., State College, Pa.) was used. The column was nonflow conditioned at 310 "C followed by an injection of Silyl-8 (Pierce Chemical Co., Rockford, 111). The JXR column thus prepared had 770 theoretical plates per foot for silylated tetracycline (TMS-TC). Reagents. The internal standard-silylation reagent. The silylation reagent was prepared as follows: 5 ml of N,O-bis-(trimethylsily1)-acetamide (BSA) (Pierce Chemical) and 5 ml of trimethylchlorosilane (TMCS, Pierce Chemical) were added to 10 ml of pyridine. Trioctanoin (Eastman Organic Chemical, Rochester, N.Y.) was then added to the silylation reagent so as to have a final concentration of approximately 1.5 ~1 per rnl of reagent. The internal standard-silylation reagent was prepared daily and was immediately used. Procedure. Reference Standard. About 10 mg of an in-house tetracycline hydrochloride reference standard was accurately weighed into a one-dram screw cap vial (Kimble Glass, OwensIllinois, Toledo, Ohio, Part. No. 60910) and sealed with a 2.6-mil polyethylene lined cap. Sample. About 10 mg of tetracycline hydrochloride powder was accurately weighed into a one-dram screw cap vial. Silylation Procedure. One milliliter of the internal standardsilylation reagent was added t o each vial containing tetracycline by use of a glass tuberculin syringe. The sample thus prepared was silylated at room temperature for over 24 hr.
RESULTS AND DISCUSSION Silylation. Formation of a n intact silylated derivative of tetracycline (TMS-TC) was quite difficult. Weaker silylation reagents than the mixture of BSA and T M C S resulted in the formation of unstable derivatives and stronger silylation reagents and their combination degraded the T C molecule. An equal proportion of BSA and TMCS in pyridine was optimal for the successful and reliable formation of TMS-TC. An increased proportion of T M C S to BSA degraded the T C and a decreased proportion of the T M C S t o BSA resulted in incomplete silylation. Silylation temperatures above 35 "C degraded T C and temperatures below 15 "C slowed the derivatization considerably without a n additional benefit. Silylation of T C is completed in 24 hr at room temperature and the TMS-TC is stable for at least 2 days in the silylation reagent when kept at or below room temperature. The TMS-TC was characterized by introducing the sample by the solid probe inlet of a n LKB 9000 GC-MS. The mass spectrum of TMS-TC (Figure 1) showed the parent ion t o be m / e 804 indicating attachment of five
r CONH, OTMS
0
TMSO
M S
0
-TMSOH
699 4
i
1 -HN(CH3)2
t
m/e 654 j-TM+to
/f, /e ;:;20 , rn/e 2 4 2
m/e 547 J-TMSOH m/e 457
m/e 714 -TMS
I t
-
m/e 642=rn/e624 /-tH3 \ I 6 2 7
m/e 626
I -to",
T
m/e 502 m/e 4 4 2
Figure 2. Possible fragmentation of penta-TMS-TC
T M S t o the T C molecule (Figure 2). An evaluation of the mass spectrum for possible fragmentation patterns of the penta-TMS-TC indicated a loss of m / e 44 from the parent ion. This indicated a loss of either a n unsilylated -CONH2 or -N(CH3)2. The m / e 547 and 242 may be the fragments of demethylated penta-TMS-0-TC ( m / e 789) cleaved at 4-4' and 12'-1 linkages of the D ring. The molecular model (Dreiding Stereomedel, W. Buchi Glasapparatefabrik, Switzerland) of T C indicated t h a t the O H group at t h e 12' position is not structurally hindered and the silylation condition used favors silylation of a n unhindered OH group more than an amide group (27). An attempt t o completely silylate T C using TMSDEA, a reagent frequently used to silylate an amine group ( 2 4 ) , re(27) A. E. Pierce, "Silylation of Organic Compounds," Pierce Chemical Co., Rockford. Ill., 1968.
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 12, OCTOBER 1973
2137
Table I . Relative Retention of TMS-Tetracyclines from the Internal Standard on Various Stationary Phasesa Internal standard: trioctanoin 3% JXR
3% o v - 1 TMS derivatives of
Doxycycline Oxytetracycline Tetracycline 4-epi-Tetracycline Anhydrotetracycline 4-epi-Anhydrotetracycline
Chlorotetracycline Theoretical plates per f t for TMS-TC
3% SE-52
(methyl
(methyl
(5% phenyl silicone) 2.60
3% o v - 1 7 (50% phenyl
1 0 % OV-25 (75% phenyl
silicone) 2.18
silicone) 2.65 3.91 4.59 5.29 (1.15) 4.19 (0.93) 5.47 (1.19) 6.23
silicone) 2.63 3.78 4.37 4.80 (1.09) 4.00 (0.92) 5.41 (1.24) 5.65
3.83 4.71 5.52 (1.17) 4.40 (0.93) 6.15 (1.31) 6.38
4.93 5.93 ( 1 . 2 0 ) 6.32 (1.28) 8.14 (1.65) 6.66
silicone) 2.38 3.90 4.94 5.76 ( 1 . 1 7 ) 6.76 (1.37) 9.76 (1.98) 6.29
343
770
336
38 1
395
3.48
10% o v - 2 1 0
(trifluoropropyl silicone)
3.59 5.41 6.29 6.98 (1.10) 7.23 (1.15) 8 . 2 7 (1.31) 8.27 41 1
Figures in parentheses indicate separation factor from TC (ETCjTC etc.).
I
I I
L
and favored trimethylsilylation of active hydrogens of OH groups. Therefore, the penta-TMS-TC obtained is most likely penta-TMS-0-TC. The mass spectrum of TMSETC is essentially similar to that of TMS-TC. The mass spectrum of TMS-OTC is inconclusive. Separation of Tetracyclines. The separation characteristics of TMS-TC and its entities on columns packed with six different stationary phases are shown in Table I. The figures in parentheses are the separation factors between silylated derivatives of TC, ETC, ATC, and EATC. On the methyl silicone column (OV-1 and JXR), separation factors between TC, ETC, ATC, and EATC are small. However, because of the high efficiency of the JXR column (theoretical plates of 770/ft), the chromatographic separation of TC, ETC, ATC, and EATC was possible (Figure 3). Although doxycycline, oxytetracycline, and chlorotetracycline can be chromatographed on the J X R column (Figure 4), the separation of chlorotetracycline (CTC) from EATC may not always be made. For the precise differentiation of CTC from EATC the use of the OV-25 column is required. The performance of several JXR columns prepared was similar and the J X R column was the only one which made the chromatography of TC on a 6-ft long column possible. Use of a longer column than 2 ft for the other stationary phases was not successful due mostly to the high operating temperature required, which resulted in a marked increase of on-column adsorption and degradation of TMS-TC. Separation of ETC from ATC was poor on the SE-52, OV-17, and the OV-210 columns. Although separation factors between TC and its entities were excellent on an OV-25 column, reproducibility of the separation by the identically packed column was poor and the column life was short. The on-column adsorption of TMS-TC was also high and quantitation was poor (relative standard deviation of over 5%). Quantitative Determination of Tetracycline. The peak height ratio between TC and the internal standard against weight of TC was linear over the range of 4 to 16 mg TC hydrochloride (y = 0 . 1 1 6 ~- 0.187) with a standard deviation of 0.022. The precision of the gas chromatographic assay method was determined by comparing ten replicate preparations of one tetracycline hydrochloride powder. Table I1 indicates that the relative standard deviation of the TC determination is 2.3%. A typical chromatogram of a commercially obtained TC hydrochloride powder may be seen in Figure 5. Twenty-six lots of tetracycline hydrochloride powder from various commercial sources together with the USP and the Upjohn in-house TC reference standards were analyzed for potency by the UV and the microbiological cylinder cup methods using Bacillus cereus (ATCC 9634) and for both potency and composition by the GLC method
~a, 3
I
\
0
4
8
12
16
20
28 32 MINUTES
24
36 40
-
4
I
44
"
48
52
56
Figure 3. Isothermal separation of tetracycline 4-epitetracycline anhydrotetracycline and 4-epianhydrotetracycline at 245 "C using a 6-ft 3% JXR column ( 1 ) ATC (2) TC (3) ETC (4) EATC
1
I
MINUTES
Figure 4. Composite chromatogram of doxycycline oxytetracycline, and chlorotetracycline r u n isothermally at 260 "C using a 6-ft 3% JXR column (1) Internal standard, (2) doxycycllne; (3) oxytetracycllne; (4) chlorotetracycline, and (5)declomyctne
sulted in GC peaks of longer retention time than the penta-TMS-TC indicating inconsistent formation of hexaTMS-TC. The mass spectrum did not support enolization 2138
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 1 2 , OCTOBER 1973
Table II. Precision of the Tetracycline Gas Chromatographic Assay Method by Peak Height Ratio Wt of tetracycline, mg
lnternai standard height
10.050 10.060 10.068 10.140 10.000 10.100 10.120 9.934 10.158 10.096
33.8 34.0 34.7 29.4 28.9 31.8 36.2 26.6 39.0 31.7
Tetracycline height
47.3 44.8 47.6 40.5 38.4 42.7 50.2 34.7 54.1 44.7 Relative standard deviation
1
Height ratio/wt
0.1392 0.1310 0.1362 0.1359 0.1329 0.1329 0.1 370 0.1313 0.1 366 0.1397 2.28%
(Table III). The potencies obtained by the three methods were then compared using analysis of variance. The results of this analysis are listed in Table IV. Therefore, it can be concluded that there is no significant difference in these three assay methods. The per cent composition of ETC, ATC, and EATC in 26 commercially purchased TC powders was determined by GLC (Table III). The averaged results are as follows: ETC 2.670, ATC 1.4%, and EATC/CTC 0.2%. The analysis of TC samples stored 6 or more years at room temperature showed the following results: ETC 4.770, ATC 2.4%, and EATC/CTC 0.8% (Table V). The slight increase in degradation compounds shown may be due to the degra-
0
4
11 8
12 MINUTES
16
20
24
Figure 5. A typical chromatogram of tetracycline r u n isothermally at 260 "C using a 6-ft 3% JXR column (1) Internal standard: (2) TC
dation of the TC samples during storage and/or to the differences in production processes and sources of the samples.
Table I I I. Tetracycline Hydrochloride Powder from Various Suppliers Supplier
Lot No.
USP Reference Standard
Microbiological Assay, Pglw
Composition of various tetracycline entities % UV Assay Pglmg
GLC,
KlImg
TC
ETC
ATC
EATC/CTC
1000
989
1007
95.8
3.0
1.2
0
1000
1000
1000
95.1
2.7
1.7
0.5
984 967 936 98 1 980 996 987 995 992 995 998 1009 952 974 920 977 1013 971 990 969 977 998 995 968 962 984
969 988 990 9 79 983 971 973 972 965 977 968 967 993 982 988 982 970 967 969 967 9 70 969 980 984 984 972
987.5 952.6 985.5 1022 991.4 972.7 986.8 978.6 992.6 977.9 994.9 998.9 941.5 990.3 968.5 1007 1023 991.2 990.5 980.3 995.3 976.1 972.6 996.5 984.7 952.5
94.5 95.1 95.0 94.2 95.3 96.4 96.6 96.8 94.9 95.9 96.1 94.2 96.8 97.3 95.4 96.2 97.1 97.2 96.3 94.4 96.5 96.8 94.7 96.1 94.7 94.8
3.3 2.9 3.0 3.2 2.6 2.2 2.1 1.9 3.3 2.9 2.3 3.2 2.0 1.6 3.0 2.6 1.7 1.5 2.2 3.5 2.5 2.0 3.0 2.6 3.9 4.2
1.7 1.5 2.0 2.0 1.6 1.4 1.3 1.3 1.8 1.2 1.6 2.0 1.2 1.1 1.6 1.2 1.2 1.3 1.5 2.1 1 .o 1.2 1.8 1.3 0.9 0.5
0.5 0.5 0 0.6 0.5 0 0 0 0 0
0 0.6 0 0 0 0 0 0 0.6 0 0 0 0.5 0 0.5 0.7
95.72
2.68
1.44
0.20
U p j o h n in-house
reference standarda Upjohn laboratory standard A
B
C
D
E
F G
1 2 3 4 5 6 7 8 1 2 3 1 2 3 4 1 2 3 4 5 6 1 2 1 1
Average a
980.4b
Used as the standard to calculate potencies.
976.66
985.gb
Value excluding the in-house standard
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 12, OCTOBER 1973
2139
GLC and partition column method were examined by weighing samples of TC and ATC in the ratio of 24, 40, and 60% of ATC in TC. Purity of the TC and ATC powders used were also determined (Table VI). The percentage of TC and ATC in each of the three mixtures was then corrected for purity. The results were evaluated by linear regression and indicate that recovery of ATC by both the GLC and the column methods was linear over
Table IV. Statistical Analysis of TC Potency between Microbiological, UV, and GLC Methods 95% confidence limit - 5 . 5 to 13.1 - 5 . 7 to 1 6 . 7 - 1 . 9 to 2 0 . 5
Difference between Microbioassay and U V
Microbioassay and GLC and U V
GLC
Table V. Tetracycline Hydrochloride Aged at Room Temperature Composition of various TC entities, % Length of storage of TC at room temperature
years, 10 months 6 years, 8 months 6 years, 6 months 6 years, 4 months 6 years, 2 months 6 years, 1 month 7
Average
Microbiological assay. Ccgimg 986
G LC
990 944 964 956 965
UV assay, !-lg/mg 964 964 971 957 961 948
G LC assay, Ccglmg 999.4 979.4 976.5 987.6 977.3 939.4
TC 91.0 91.8 93.3 90.7 92.5 93.7
ETC 5.1 4.2 4.0 5.9 4.5 4.3
ATC 2.8 3.2 2.1 2.1 2.4 2.0
EATC/CTC 1.1 0.8 0.6 1.3 0.7
967.5
960.8
976.6
92.2
4.67
2.43
Table VI. Analysis of TC and ATC Samples Used to Compare Sensitivity of Partition Column Chromatography and GLC Compnsition, % TC
ATC
ETC
EATC
CTC
0
0.5 9.3
0 0
4.2
0
0
2.3
0 0
G LC TC ATC
95.1
TC ATC
95.8
0
0
97.6
0
1.7 90.7
2.7
Column
The GLC determination of ETC, ATC, and EATC in aged TC samples was then compared with the modified diatomaceous earth column method (18) of Ascione et al. (12) (Table V). Although the ETC value obtained by the two methods showed no significant difference, the GLC method gave significantly higher ATC and EATC values than those by the column method. The higher ATC value by the GLC method may partly be attributed to an incomplete separation of ATC from TC and/or to the detection limit of the column method (ca. 1%)(18). The detection limit of the GLC method is approximately 0.5% when the ball and disk integration method is used. The following experiment was conducted to support the above hypothesis. The differences in the ATC value by
2140
Column
0
TC 91.7 92.7 94.5 96.3 95.8 93.8
ETC 5.7 6.2 5.4 3.7 4.1 5.3
0.75
94.1
5.07
ATC
EATC/ CTC
0
trace
1.0
0
0 0 0 0
0.8
0
0.30
0
0 0
-
the range of 0 to 6O%, y = 1 . 0 4 ~- 2.71 for the GLC and y = 1 . 1 0 ~- 0.69 for the column method. Both the slope and intercept of the two methods were not significantly different. Therefore, the differences in the ATC value between the two methods shown in Table V may be caused by the differences in sensitivity of detection of ATC by the two methods. The purity of ATC as determined by the GLC method (90.7%) agreed well with that obtained (91.8%) by a high pressure liquid chromatographic method (unpublished) which can determine TC without further derivatization. Therefore, the results obtained above indicate that the silylation procedure developed did not yield detectable quantities of degradation products.
ACKNOWLEDGMENT Bristol Laboratories and Charles Pfizer and Company are acknowledged for the supply of doxycycline hyclate, oxytetracycline, chlorotetracycline, 4-epitetracycline, anhydrotetracycline, and 4-epianhydrotetracycline. Reviews of TMS-TC fragmentation patterns by K. L. Rinehart of the University of Illinois, C. C. Sweeley of Michigan State University, and P. B. Bowman and L. Baczynski of The Upjohn Company are gratefully acknowledged. A. R. Lewis is acknowledged for the statistical analysis. Received for review March 5 , 1973. Accepted April 6, 1973.
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 12, OCTOBER 1 9 7 3
