Anhydrotetracycline and 4-Epianhydrotetracycline in
`Market Tetracyclines and Aged Tetracycline Products
`
`VERNON C. WALTON, MARGARET R. HOWLETT, and GEORGE B. SELZER
`
`Abstract 0 A large number of tetracycline samples were tested for
`the presence of anhydrotetracycline and 4-epianhydrotetracycline
`when they were fresh and after being stored under normal and ad-
`verse conditions. It was found that: (a) Newly manufactured tetra-
`cycline preparations contain only small amounts of anhydrotetra-
`cycline and 4-epianhydrotetracycline. (b) Storage under adverse
`conditions markedly increases the percentage of degradation
`products, but storage under normal conditions results in only a
`slow increase in anhydrotetracycline and 4-epianhydrotetracycline.
`In syrups, this can be correlated with loss in tetracycline potency.
`(c) Citric acid greatly increases the tendency of tetracycline to de-
`grade. (4 Tetracycline hydrochloride is more stable than tetra-
`cycline phosphate. (e) Demethylchlortetracycline is the most
`stable of the tetracycline derivatives studied. (f) Rolitetracycline is
`extremely unstable.
`Keyphrases 0 Anhydrotetracycline determination-tetracycline
`products [3 4-Epianhydrotetracycline determination-tetracycline
`products 0 Column chromatography-separation 0 UV spectro-
`IJ
`photometry-analysis
`Colorimetric analysis-spectrophotom-
`eter 0 Turbidimetric analysis-biological potency
`
`Degradation products of tetracycline (TC) and 4-
`epianhydrotetracycline (EATC), in particular, have been
`implicated in renal dysfunction (1-9). It was, therefore,
`of interest to determine the amount of degradation in
`TC products on the market. A large number of market
`TC products were examined for EATC content. Several
`lots were tested for stability under normal and adverse
`storage conditions to determine the amount of EATC
`that may reasonably be expected in newly manufactured
`TC products and to reveal what increase in EATC could
`be expected with time.
`Several analytical methods for the determination of 4-
`epitetracycline (ETC), anhydrotetracycline (ATC), and
`EATC have been described (10-17). The most conve-
`nient are those of Dijkhuis (15), Kelly (16), and Selzer
`and Wrisht (17); these have been adopted for use in this
`study.
`
`EXPERIMENTAL
`
`Methods-Colrrtm Chromatdgraphy-The method of Kelly (16)
`is a column chromatographic separation of ATC and EATC from
`each other and from TC and other interfering substances. The
`column consists of acid-washed diatomaceous earth' moistened
`with 0.1 M ethylenediaminetetraacetic acid (EDTA) buffer, pH
`7.8; the compounds are eluted with chloroform and determined
`spectrophotometrically. For this study, the method was modified
`to ensure that the sample was at pH 7.8 when it was applied to the
`column. A 5-ml. sample of TC syrup containing 25 mg. of TC/ml.
`or a 2-ml. sample of pediatric drops containing 100 mg. of TC/ml.
`was diluted to 10 ml. with 0.1 MEDTA buffer, pH 7.8 (prepared by
`dissolving 0.1 mole of EDTA disodium salt in 800 ml. of water,
`adjusting the pH to 7.8 with ammonium hydroxide, and diluting to
`1 1.). The sample was then brought to pH 7.8 with ammonium
`
`1 Celite 545, Johns-Manville, New York, N.Y.
`
`1160 IJ Journal of Pharmaceutical Sciences
`
`Table I-Total Anhydrotetracyclines Present in Fresh
`Tetracycline Powder"
`
`Sample
`Number
`
`Manufacturer
`
`1-4
`5-8
`9-12
`13-18
`19-2 1
`22-25
`26-29
`3&33
`34
`35
`36-38
`
`A
`B
`C
`D
`E
`F
`G
`I
`J
`K
`L
`
`ATC + EATC, Z
`
`0.17,O. 14, 0.31, 0.17
`0.97.0.86.0.90.0.92
`0.47; 0.48; 0.57; 0.98
`2.06, 1.44, 1.98, 1.59
`1.69, 1.27
`0.25,0.71,0.62
`0.24,0.25,0.23,0.21
`0.39,0.46,0.36,0.34
`0.20,0.17,0.66,0.06
`0.11
`0.25
`0.41,0.09,0.81
`
`a Determined by the method of Dijkhuis (15).
`
`hydroxidewater (1 : 9) and diluted to 25 ml. with the EDTA buffer.
`One tablet containing 125 mg. of TC or capsule material containing
`125 mg. of TC was ground in a mortar and pestle with 10 ml. of 0.1
`M EDTA buffer, pH 7.8, brought to pH 7.8 with ammonium hy-
`droxidewater (1:9), and diluted to 25 ml. with the EDTA buffer.
`Each 250-mg. capsule was blended with 25 ml of 0.1 M EDTA
`buffer, pH 7.8, brought to pH 7.8 with ammonium hydroxide-water
`(1 : 9), and diluted to 50 ml. with the EDTA buffer. Difficultly soluble
`samples were dissolved first in 10 ml. of 0.1 N HC1, brought to pH
`7.8 with ammonium hydroxidewater (1 : 9), and then diluted to 50
`ml. with 0.1 MEDTA buffer, pH 7.8.
`A 1-ml. aliquot of the diluted sample was then mixed with 1 g.
`of dry diatomaceous earth, applied to the column, and covered with
`a 1-cm. layer of diatomaceous earth moistened with 0.1 M EDTA
`buffer, pH 7.8. The method was further modified in that no sup-
`porting circle of filter paper was used under the column, no layer
`of sand was used, and the 0.1 M EDTA buffer was not equilibrated
`with CHCb before use.
`Spectrophotometric Screening Method-The method of Dijkhuis
`total anhydrotetracycline content (ATC + EATC). The sample
`(15) was used to screen powder, tablet, and capsule samples for
`preparation was modified to ensure the dissolution of samples con-
`taining TC base or TC phosphate. A 50-mg. sample of TC powder
`was weighed into a SO-ml. volumetric flask, and 10 ml. of 0.1 N HCI
`was added. The flask was shaken until the sample dissolved; the
`contents were then diluted to the mark with water and assayed.
`Capsule powder or finely ground tablet powder equivalent to 250
`mg. of TC hydrochloride was placed in a 250-ml. volumetric flask;
`50 ml. of 0.1 N HC1 was added, and the flask was shaken on a
`mechanical shaker for 5 min. The sample was then diluted to
`volume with water and filtered through a fluted-filter paper. The
`fist 20 ml. of filtrate was discarded and the remainder was collected
`for assay. The calculations were modified in that the formula recom-
`mended by Dijkhuis (15) for the calculation of the percent ATC
`in dosage forms was also used for TC powder.
`Paper Chrontatography-The method of Selzer and Wright (17)
`was used. It is an ascending paper chromatographic method for the
`separation of TC compounds by a resolving solvent of chloroform-
`nitromethane-pyridine (10:20:3) on 20.3 x 20.3-cm. (8 X 8-in.)
`Whatman No. 1 paper moistened with McIlvaine's buffer, pH 3.5;
`the spots are observed by their fluorescence under UV light. To
`vary the relative positions of the different TC and degradation
`products on the chromatogram and to facilitate their identification,
`the chromatographic paper was moistened with any of three buffers:
`McIlvaine's buffer, pH 3.5; 0.1 M EDTA buffer, pH 4.5; or 0.1 A4
`EDTA buffer, pH 7.7.
`
`Merck Exhibit 2248, Page 1
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`Sample Number
`
`Manufacturer
`
`~~~
`
`Table 11-Total Anhydrotetracyclines Present in Fresh
`Tetracycline Phosphate Powder"
`ATC + EATC, %
`1.44
`1.58
`1.05
`1.07
`1.05
`
`1
`2
`3
`4
`5
`
`H
`H
`H
`H
`H
`
`a Determined by the method of Dijkhuis (15).
`
`Potency Determination-The biological potencies of the samples
`of TC used in these studies were determined by the turbidimetric
`assay method (18) with Staphylococcus aureus (ATCC 6538P) as the
`test organism.
`standard mixture of 50: 50
`Standards-ATC-EATC Mixture-A
`ATC-EATC was prepared as follows: a mixture of 2 g. of TC HCI,
`1 ml. of glacial acetic acid, and 20 ml. of water was incubated over-
`night at 37". One milliliter of concentrated HCl was added, the
`mixture was heated on a steam bath for 30 min., and the liquids
`were removed by lyophilization. The dry powder was dissolved in
`methanol, and an aliquot was chromatographed on paper as
`described previously, using each of the three buffers mentioned to
`moisten the paper. In each case, two spots of equal intensity, cor-
`responding to ATC and EATC, were detected. Analysis of the
`powder by the column chromatographic method indicated that it
`contained 48.9% ATC and 50.9% EATC. Comparison of the 100.
`Mc.p.s. NMR spectrum of a CF2COOH solution with data in the
`literature (19) confirmed the identity of the mixture. The bands
`in a 100-Mc.p.s. spectrum were better resolved, and those bands due
`to H, and Ha* (Structures I and 11) could be integrated. Comparison
`
`anhydrotetracycline
`I
`
`4.epianhydrotetracycline
`11
`of the peak areas indicated that the powder contained 45% ATC
`and 55% EATC.
`Additional quantities of mixed ATC and EATC were prepared
`by heating TC HCl for 5 min. in 2 N HCl(l5). The compositions of
`these materials were determined by the method of Kelly (16) and by
`comparison of their absorptivities at 356 and 430 mp.
`Anhydrotetracycline HCI Hydrate-Anhydrotetracycline HCl
`hydrate (Lot No. 63 F 2052, Bristol Laboratories, Syracuse, N.Y.)
`was used. Paper chromatography in all three buffer systems indi-
`
`Sample
`Number
`1-15
`
`Manufacturer
`F
`
`Table 111-Total Anhydrotetracycline and 4-Epianhydrotetracycline
`Present in Fresh Tetracycline Hydrochloride Tablets"
`ATC + EATC, %
`0.50,0.46,0.53,0.52,
`0.54,0.46,0.47,0.63,
`0.59,0.59,0.57,0.58,
`0.63.0.49.0.54
`i.81; i.621 1.45, 1.59
`0.14,O. 11,0.69
`
`16-19
`B
`2&22
`G
`0 Determined by the method of Dijkhuis (15).
`
`Table IV-Total Anhydrotetracycline and 4-Epianhydrotetracycline
`Present in Tetracycline Capsules"
`
`Sample
`Typeof
`Number Tetracycline
`
`Manu-
`facturer
`
`1-3
`4-5
`6-15
`
`1618
`~~ 19
`20
`21-24
`25-28
`29-31
`32-33
`34
`35-38
`39
`4049
`
`HCl
`HCI
`PO4
`
`Po4
`HCl
`Base
`HCl
`HCl
`HCI
`HC1
`HCI
`HCI
`HCl
`HCI
`
`B
`G
`H
`
`M
`M
`N
`N
`0
`P
`
`3
`
`S
`T
`U
`
`ATC + EATC, %
`1.38, 1.28,0.83
`0.29,O. 27
`1.73, 1.66, 1.62, 1.50
`1.76. 1.44.2.89. 3.05
`1.13: 1.14'
`0.60, 1.32
`1.58;
`0.57
`0.65
`0.46,0.44
`0.57,
`0.50,
`0.12,o. 55
`0.23.
`0.32,
`0.21:
`0.29,
`0.24
`0.68
`0.85;
`0.33
`0.71,
`0.63,
`0.71,0.34
`0.68
`1.29, 1.35
`1.14,
`1.09,
`1.45,
`1.41, 1.24
`1.60,
`
`1.31 1.31,
`
`a Determined by the method of Dijkhuis (15).
`
`~~~~~
`
`cated that the sample contained only a trace of EATC; none could
`be detected by column chromatography. Integration of the bands
`corresponding to protons H, and H.1 (Structures I and 11) in the
`100-Mc.p.s. NMR spectrum of a DMSO-d6 solution of the sample
`indicated that the amount of ATC was on the order of 23 times as
`much as the amount of EATC.
`4-Epianhydroretracycline Sulfate-4Epianhydrotetracycline sul-
`fate (Lot No. 57 F 536, Bristol Laboratories) was used. Paper chro-
`matography of this compound in all three systems indicated that
`only a trace of ATC was present; none could be detected by column
`chromatography. Integration of the bands corresponding to protons
`H. and Ha1 (Structures I and 11) in the 100-Mc.p.s. NMR spectrum
`of a DMSO-ds solution of the sample indicated that the amount of
`EATC was on the order of 10 times as much as the amount of ATC.
`However, the presence of traces of one or more other compounds
`was indicated.
`of the TC samples used in this study had been
`Samples-All
`received as part of the antibiotic certification program.
`
`RESULTS AND DISCUSSION
`
`Because pharmaceutical dosage forms of TC contain a number of
`other ingredients, the column chromatographic procedure and the
`screening method were tested for interference from such materials.
`Phenyltoloxamine, aspirin, phenacetin, caffeine, salicylamide, and
`chlorothen citrate did not interfere with either method. Ampho-
`tericin B, neomycin, nystatin, oleandomycin, and triacetyloleando-
`mycin had no detectable effect on the results of the column chro-
`matographic procedure. Although novobiocin caused the EATC
`band to follow the ATC band quite closely in the column chro-
`matographic procedure, it did not affect the results. However,
`
`Table V-Anhydrotetracycline and 4-Epianhydrotetracycline
`Found in Fresh Tetracycline Syrups"
`
`Sample
`Number
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`1 1
`12
`
`Manufacturer
`
`B
`B
`F
`H
`T
`T
`V
`V
`V
`V
`W
`0
`
`ATC, %
`0.27
`1.81
`0.06
`0.13
`0.13
`0.09
`0.39
`0.72
`0.11
`0.11
`2.93
`0.19
`
`EATC, %
`0.28
`1.12
`0.13
`0.13
`0.30
`0.26
`0.43
`0.70
`0.16
`0.12
`4.18
`0.47
`
`= Determined by the method of Kelly (16).
`
`~
`
`~
`
`~~~~
`
`Vol. 59, No. 8, August 1970
`
`1161
`
`Merck Exhibit 2248, Page 2
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`Table VI-Comparison of the Loss of Potency of Tetracycline
`Syrup after Storage" with the Amount of Anhydrotetracycline
`and 4-Epianhydrotetracyclines
`
`Sample
`Number
`
`Potency Lost, ATC Found,
`mg./dose
`mg./dose
`
`EATC Found,
`mg./dose
`
`1
`2
`3
`A
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16*
`17
`18
`19
`20
`21
`
`0
`0
`0
`0
`2
`5
`5
`9
`10
`11
`12
`13
`13
`16
`17
`17
`23
`25
`~~ 28
`61
`32
`
`0.16
`0.26
`0.38
`3.7
`0.49
`0.53
`0.98
`. ..
`3.5
`0.35
`0.49
`1.2
`0.32
`0.38
`4.8
`0.44
`1.4
`1.2
`2.0
`~.
`2.0
`3.6
`5.3
`
`a Storage ranged from 12 to 20 months at 25".
`formula.
`
`0.25
`0.52
`0.30
`4.6
`0.62
`0.32
`i.29
`3.9
`0.50
`0.62
`0.72
`0.32
`0.59
`4.1
`0.82
`1.5
`0.99
`2.6
`2.1
`4.1
`5.1
`b An experimental
`
`amphotericin B and nystatin, both of which absorb light at 430 mp,
`caused unduly high results in the screening method when they were
`present in concentrations comparable to those found in commercial
`products. For this reason, TC products containing amphotericin B
`or nystatin were tested by the column chromatographic method.
`were present in fresh TC powder. As shown in Table I, the ATC +
`A survey was conducted to find out how much ATC and EATC
`EATC content of 38 samples of fresh TC HC1 bulk material from
`six results over 1 z were obtained with material from one manu-
`11 manufacturers ranged from 0.11 to 2.06%. However, all of the
`facturer. Table I1 shows the total ATC + EATC found in newly
`All of the five samples tested contained between 1 and 2 z ATC +
`manufactured TC PO4 analyzed by the method of Dijkhuis (15).
`EATC.
`Another survey was made of the ATC and EATC present in
`newly manufactured samples of various TC dosage forms. Table 111
`total ATC + EATC. These five samples were produced
`shows that of 22 TC HCl tablets tested, only five contained more
`by one manufacturer and contained less than 2% ATC + EATC.
`than 1
`tained less than 2 x total ATC + EATC. Table V shows that only
`As shown in Table IV, all but two of the 49 TC capsules tested con-
`
`two of the 12 fresh TC syrups tested contained more than 1% of
`either ATC or EATC.
`It can thus be seen that the amounts of ATC and EATC found
`in fresh TC products were quite low. On the basis of these results,
`limits of 2% EATC in TC powders and 3% EATC in finished TC
`products have been proposed for inclusion in the Code of Federal
`Regulations (20).
`Several studies were conducted to determine the stability of TC
`products under various conditions. In one study, TC syrups were
`examined for ATC and EATC after they had been stored for dif-
`ferent periods of time. The solid matter in the syrups was either in
`suspension or easily resuspendable. As shown in Table VI, there is
`some correlation between loss of biological potency and increase in
`ATC and EATC content.
`In another study, the ability of the TC antibiotics to resist un-
`favorable storage conditions of elevated temperature and high
`humidity was investigated. The TC samples were stored at 37" in
`desiccators containing water (100% relative humidity) or a saturated
`sodium bromide solution (66% relative humidity) in the desiccant
`chamber. The samples were tested for potency by the microbiologi-
`cal turbidimetric method (18) and for degradation products by the
`paper chromatographic method on paper moistened with McIl-
`vaine's buffer, pH 3.5, and by the column chromatographic method.
`Several conclusions may be drawn from the data presented in
`Tables VII and VIII. Demethylchlortetracycline (DMCTC) is the
`most stable of the TC derivatives when stored under 100% relative
`humidity at 37"; it showed no loss of potency after storage for 1
`month. Chlortetracycline (CTC) is somewhat less stable than
`DMCTC; after 1 month at 37" and 100% relative humidity, the
`potency of CTC powder had diminished by 17 % and that of capsule
`material by 1 4 z . Neither CTC nor DMCXC showed evidence of
`anhydrolike degradation products by the paper chromatographic
`method. However, TC was less stable; TC products stored 1 month
`at 37" and 100% relative humidity gave visible evidence of degrada-
`tion by becoming partially liquid and turning dark brown. Potency
`losses of from 17 to 7 9 x occurred, and the column chromato-
`graphic procedure revealed ATC levels ranging from 2.2 to 7.1 Z
`and EATC levels ranging from 3.3 to 14.1 %. Paper chromatography
`indicated that approximately one-half of the TC was converted to
`the inactive epimer form, ETC. Rolitetracycline (RTC) was even
`more unstable. Storage at 37" and 100% relative humidity resulted
`in almost complete destruction of this antibiotic; the only recog-
`nizable fragment was a small amount of the epimeric form of RTC.
`Paper chromatography of degraded oxytetracycline indicated the
`possible presence of an anhydrolike product.
`TC HCI powder and dosage forms were quite stable when stored
`2 months at 66% relative humidity and 37", as shown in Table IX.
`Only a small fraction of the TC was converted to the anhydro forms.
`However, TC HCI with added citric acid was almost completely
`inactivated and formed large amounts of ATC and EATC. Citric
`acid thus has an adverse effect on TC stability. TC POa is somewhat
`
`Table VII-Results of the Storage of Tetracycline Derivatives for 30 Days
`at 37" and 100% Relative Humidity
`
`Product
`
`Pooled demethylchlortetracycline
`hydrochloride powder
`Demethylchlortetracycline
`capsule powder
`Oxytetracycline hydrochloride
`powder
`Oxytetracycline base powder
`
`Rolitetracycline powder
`
`Description
`after Storage
`
`Brown powder
`Light-brown
`powder
`Dark-brown
`Dark-brown liquid
`with few solid
`particles
`Very dark-brown
`tar
`
`Yellow powder
`
`Chlortetracycline hydrochloride
`powder
`Chlortetracycline capsule
`powder
`a On paper moistened with McIlvaine's buffer, pH 3.5.
`1162 0 Journal of Pharmaceutical Sciences
`
`-Potency, mcg./mg.-
`Before
`After
`Storage
`Storage
`
`1000
`490
`
`1000
`
`I000
`
`490
`
`1030
`480
`
`490
`185
`
`15
`
`420
`
`Paper Chromatography"
`
`Strong fluorescent spot (DMCTC)
`at RJ 0.63
`Two minor spots at R, 0.19 (Epi-
`DMCTC) and Rj 0.37
`Strong fluorescent spot (OTC)
`at RJ 0.20
`Strong blue-green fluorescent
`spot at origin; RJ 0.39 and 0.96
`
`No TC detected. Weak fluorescent
`spots at RJ 0.38, 0.20, and 0.96.
`Strong spot at origin.
`Strong fluorescent spot (CTC) at
`RJ 0.74
`Weak TC spot at R, 0.43. No
`degradation indicated
`
`Merck Exhibit 2248, Page 3
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`Table VLII-Results of the Storage of Tetracycline Powder and Capsule Materials for 30 Days at
`37" and 100% Relative Humidity"
`
`Product
`
`pH before
`Storage
`
`Description after Storage
`
`-Potency, mcg./mg.-
`Before
`After
`Storage
`Storage
`
`ATC,
`
`TC PO4 powder
`TC HCI powder
`TC HCI with glucosamine
`(capsule powder)
`TC PO4 capsule powder
`(Company H)
`TC PO4 capsule powder
`(Company M)
`
`2.38
`2.30
`
`2.58
`
`2.46
`
`Very dark-brown
`viscous liquid
`Dark- brown
`powder
`Partially liquid,
`yellow- brown
`powder
`Partially liquid,
`very dark-
`brown powder
`Very dark-
`brown liquid
`
`800
`550
`520
`
`620
`
`5 70
`
`215
`350
`430
`
`130
`
`160
`
`2.2
`2.2
`7. I
`
`2.3
`
`5.7
`
`EATC, X,
`3.3
`3.7
`6.5
`
`5.2
`
`14.1
`
`a Chromatography was done on paper moistened with McIlvaine's buffer, pH 3.5; strong fluorescent spots for TC at Rr 0.46 and Epi-TC at Rj
`0.14; spots for ATC at Rf 0.98 and EATC at R f 0.60.
`
`Table IX-Results of Storage of Tetracycline Powder and Capsule Materials at 37"
`and 66% Relative Humidity
`
`Product
`
`Days
`Stored
`
`Description
`after Storage
`
`Potency, mcg./mg. Loss in
`Before
`After Potency,
`Storage Storage
`
`ATC, % EATC, %
`
`TC HC1
`TC HCI capsule powder
`TC HCI with citric acid
`capsule powder
`TC PO4 powder
`
`TC PO4 capsule powder
`(Company H)
`TC PO4 capsule powder
`(Company M)
`
`70
`
`70
`70
`62
`
`62
`
`62
`
`Yellow-brown
`powder
`Yellow-brown
`powder
`Orange-brown
`cake
`Yellow-brown
`powder
`Yellow-brown
`powder
`Yellow-brown
`powder
`
`1000
`(est.)
`520
`480
`800
`
`680
`
`600
`
`900
`600
`35
`480
`
`450
`
`450
`
`10
`0
`93
`40
`
`34
`
`25
`
`0.02
`0.15
`18.5
`2 . 0
`
`1.3
`
`1.7
`
`0.2
`None
`56.7
`3.2
`
`1.8
`
`2.3
`
`Paper Chromatography"
`No ATC or EATC detected
`No ATC or EATC detected
`Large amount of ATC and
`EATC. No TC detected
`Spots for ATC and EATC
`visible; large TC and
`ETC spots
`Spots for ATC and EATC
`visible; large TC and
`ETC spots
`Spots for ATC and EATC
`visible; large TC and
`ETC spots
`
`a On paper moistened with McIlvaine's buffer, pH 3.5.
`
`less stable than TC HCI under conditions of 37" and 6 6 z relative
`humidity; TC POc products stored under these conditions had
`potency losses ranging from 25 to 40% and small amounts of ATC
`and EATC were present.
`The effects of other active ingredients on the stability of TC PO1
`were also investigated. Samples from two lots of TC PO4 capsules
`made by the same manufacturer were stored under adverse and
`control conditions for 3 years. One lot (A) contained TC POc and no
`other active ingredients; the other lot (B) contained TC PO4, phenyl-
`
`Table X-Percent of Labeled Potency Present as
`Anhydrotetracycline and Epianhydrotetracycline in Tetracycline
`Phosphate Capsule Powder Stored under Normal Conditions
`and at 32 O and 63 % Relative Humidity
`32" and 63 R.H.
`Months
`Normal Conditions
`ATC, %
`EATC, %
`EATC, % ATC,
`Stored
`
`0
`4
`9
`32
`
`0
`4
`9
`32
`
`0.52
`0.8
`1.4
`3.9
`
`1 . 5
`2.3
`2.6
`2.8
`5.1
`
`Lot Aa
`0.27
`0.3
`0.88
`2.9
`Lot Bb
`0.44
`0.6
`1.2
`1 . 1
`3.7
`
`2.0
`3.3
`8.6
`
`2.7
`5.7
`3.5
`13.0
`
`2.6
`3.4
`10.6
`
`2.2
`3.7
`4 . 0
`22.1
`
`5 No other active ingredients present. b Contained phenyltoloxamine
`citrate, aspirin, phenacetin, and caffeine.
`
`toloxamine citrate, aspirin, phenacetin, and caffeine. Control cap-
`sules were stored at room temperature in closed bottles, and the test
`capsules were stored under adverse conditions in open beakers in a
`desiccator at 32". The desiccant chamber was filled with a saturated
`solution of NasCr207 to ensure a constant relative humidity of 63 %.
`At intervals the capsules were tested for ATC and EATC content.
`As shown in Table X, the test capsules of both lots at all times con-
`tained greater amounts of ATC and EATC than the control cap-
`sules; and at all times, Lot B contained greater amounts of ATC and
`EATC than did Lot A. The final potency of Lot A after being stored
`under adverse conditions was 24.4% of the labeled potency; after
`being stored under control conditions, it was 80x of labeled po-
`tency. The final potency of Lot B after being stored under adverse
`conditions was 13.4z of labeled potency; under control conditions,
`it was 85 %. The combination of phenyltoloxamine citrate, aspirin,
`phenacetin, and caffeine apparently had some additional deteriorat-
`ing effect on the stability of TC PO4 capsules stored under adverse
`conditions.
`
`REFERENCES
`
`(1) J. M. Gross, Ann. Intern. Med., 58,523(1963).
`(2) G. W. Frimpter, A. E. Timpanelli, W. J. Eisenmenger, H. S.
`Stein, and L. I. Erlich, J. Amer. Med. Ass., 184, 11 l(1963).
`(3) S. R. Sulkowski and J. R. Haserick, ibid., 189,152(1964).
`(4) W. W. Cleveland, W. C. Adams, and J. B. Man, J. Pediat.,
`66,333(1965).
`(5) N. Fulop and A. Drapkin, New EngI. J. Med., 272,986(1965).
`(6) J. Brodehl, K. Gellissen, W. Hogge, and H. Schumacher,
`Helu. Paediat. Acta, 23,373(1968).
`(7) K. F. Benitz and H. F. Diermeir, Proc. SOC. Exp. Biof. Med.,
`115,930(1964).
`
`Vol. 59, No. 8, August 1970 0 1163
`
`Merck Exhibit 2248, Page 4
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`(8) R. R. Lindquist and F. X. Fellers, Lab. Invest., 15,864(1966).
`(9) M. B. Lowe and E. Tapp, Arch. Pathol., 81,362(1966).
`(10) L. Rustice and M. Ferappi, Boll. Chim. Farm., 104, 305
`(1967).
`(11) D. L. Simmons, C. M. Koorengevel, R. Kubelka, and P.
`Seers, J. Pharm. Sci., 55, 219(1966).
`(12) D. L. Simmons, H. S. L. Woo, C. M. Koorengeval, and P.
`Seers, ibid., 55, 1313(1966).
`(13) B. W. Griffiths, ibid., 55, 353(1966).
`(14) A. A. Fernandez, V. T. Noceda, and E. S. Carrera, ibid.,
`58, 443(1969).
`(15) I. C. Dijkhuis, Pharm. Weekbl., 102, 1308(1967).
`(16) R. G. Kelly, J. Pharm.Sci., 53, 1551(1964).
`(17) G. B. Selzer and W. W . Wright, Antibiot. Cliemother., 7 ,
`2921957).
`(18) "Code of Federal Regulations," Title 21, Part 141c, Section
`141~.218(a).
`
`(19) M. Schach von Wittinau and R. K. Blackwood, J. Org.
`Chem., 31,613(1966).
`(20) Fed. Regist., 34,12286 (1969).
`
`ACKNOWLEDGMENTS AND ADDRESSES
`
`Received January 13, 1970, from the National Center for Anti-
`biotics and Insulin Analysis, Office of Pharmaceutical Sciences,
`Food and Drug Administration, Department of Health, Education,
`and Welfare, Washington, DC 20204
`Accepted for publication March 4, 1970.
`The NMR work was done by Elizabeth Hansen of the Additives
`and Instrumentation Branch of the Division of Food Chemistry
`and Technology, Bureau of Foods, Pesticides, and Product Safety.
`The potency assays were performed by the Microbiological Assay
`Branch of the National Center for Antibiotics and Insulin Analysis.
`
`NMR Analysis of Synthetic Corticosteroids of the
`1,4-Dien-3-one Type
`
`HAJRO W. AVDOVICH, PAUL HANBURY, and BRUCE A. LODGE
`
`2 .o
`
`I
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`1
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`'
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`T
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`
`1
`
`400
`
`I! L
`
`t
`
`Impurity
`
`Abstract A procedure for the analysis of synthetic corticosteroids
`of the 1,4-dien-3-one type is described. The method is based upon
`NMR spectroscopy. Spectra are determined in dimethyl sulfoxide
`containing an internal reference substance, fumaric acid. Both
`bulk drugs and formulations can be assayed using this method, and
`comparison is made with results obtained from official assays on
`the steroids and their formulations. The average deviation obtained
`in the NMR method was 0.6%. A procedure for water-soluble 1,4-
`dien-3-ones is also described. This method uses triethylamine hy-
`drochloride as an internal standard.
`Corticosteroids, 1,4-dien-3-one type-analysis 0
`Keyphrases IJ
`NMR spectroscopy-analysis 0 Fumaric acid-internal standard
`
`Synthetic corticosteroids of the lY4-dien-3-one type
`are at present assayed (1, 2) by colorimetric methods
`based on the reduction of certain tetrazolium deriva-
`tives by the a-ketol side chain at C-17. Such methods
`do not distinguish between 1,4-dien-3-ones and re-
`lated corticosteroids of the 4-en-3-one type, which
`may be present as impurities and which frequently
`possess different corticoid activity to that desired in the
`Iy4-dien-3-one. NMR spectroscopy affords a method
`of distinguishing easily between the two groups of
`steroids. 1 ,CDien-3-ones possess three vinylic protons,
`of which the chemical shift usually differs from that of
`the single vinylic proton of 4-en-3-ones. It is, therefore,
`theoretically possible to detect the two groups of com-
`pounds in the presence of each other and, hence, to
`develop a much more specific assay procedure.
`Present methods used in the determination of pred-
`nisolone sodium phosphate (PSP) (1, 2) are also rel-
`atively nonspecific. The BP method (1) for the bulk
`drug relies upon dissolution in water and measure-
`ment of the extinction of the solution at 247 mp; for
`
`1164 0 Journal of Pharmaceutical Sciences
`
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`
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`
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`
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`
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`I
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`.
`I
`.
`.
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`.
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`I
`PPM (6)
`P.0
`6.0
`7.0
`Figure 1-Partial NMR spectrum of steroidal 1,4-dieti-3-one in
`dimethyI sulfoxide containing fumaric acid and 2,5, or 10 2 of added
`steroidal 4-en-3-one.
`
`
`I
`
`.
`
`.
`
`L
`
`
`
`Merck Exhibit 2248, Page 5
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`