Does Repeated Heat Sterilization of Local
`Anesthetic Drugs Aflect Potency?
`
`L. Donald Bridenbaugh, M.D., and Daniel C. Moore, M.D.
`
`Samples of all the commonly used local anes-
`thetic drugs were autoclavcd for periods varying
`from 30 minutes to 3 hours. These were subse-
`quently assayed for potency. None of the drugs
`tested showed any appreciable loss of potency
`following autoclaving at 18 pounds pressure at
`260—275° F. for 8 hours.
`
`IN xu:cr..\"r years, theinecessity for heat steri-
`lization of
`local anesthetic drugs has been
`recognized. In some hospitals,
`if a sterilized
`ampule is not used it may be reautoclaved for
`a subsequent procedure;
`thus, some ampules
`may be subjected to several autoclavings be-
`fore use. However, as newer local anesthetic
`agents have become available, their tolerance
`to heat sterilization has not been adequately
`evaluated. The following questions may be
`raised: can all local anesthetic agents be heat
`sterilized without loss of anesthetic potency; if
`so, ho\v many times? This study was under-
`taken to answer these questions and to evaluate
`our method of heat
`sterilizaiton of
`local
`anesthetic drugs.
`
`Method of Study
`The commonly used local anesthetic agents
`were selected for testing,
`i.c., niphauoid crys-
`tals of tetracaine (Pontocaine),° and solutions
`of each of the following: procaine (Novocain),
`piperocaine (Metycaine), dibucaine (Nuper-
`caine), chloroprocaine
`(Nesacaine) mepiva-
`caine
`(Carbocaine),
`lidocaine
`(Xylocaine),
`and hexylcaine (Cyclaine).
`Six samples of
`each drug were selected and numbered 1
`through 6. The samples were subjected to
`heat sterilization in the unopened bottle or
`ampule as received from the pharmaceutical
`
`Accepted for publication December 20, 1963.
`The authors are in the Department of Anesthesi-
`ology, The Mason Clinic, Seattle, Washington.
`° Tetracaine crystals were used because this is
`the drug form used and recommended in our
`institution for regional anesthesia.
`
`372
`
`company. With the exception of one sample
`of each drug which served as a control, all
`others were placed in a regional block tray
`and autoelaved at .‘Z60—275° F. at 18 pounds
`pressure for 30 minutes. The trays were then
`cooled, one sample of each was removed and
`the time and number recorded. This process
`was repeated for each drug until
`there were
`samples which had been autoclaved for 30
`minutes, 60 minutes, 90 minutes, 120 minutes,
`and 180 minutes,
`respectively.
`This was
`equivalent to autoclaving the drug from one
`to six times under this system of sterilization.
`The numbered samples were then sent
`to
`the various drug manufacturers for assay of
`potency in a blind study.
`
`Results
`
`Procaine (2 per cent, 30 ml. vial), mepiva-
`caine (2 per cent, 50 ml. vial), and two sam-
`ples of
`tetracaiue
`(20 mg. and 250 mg.
`ampules of niphanoicl crystals) were sent to
`the manufacturer for assay. The results were
`reported as follows:
`samples were
`Procaine.
`Four milliliters
`made alkaline with 5 ml. of 10 per cent
`sodium carbonate solution and extracted 3
`times with 10-ml. volumes of
`chlorolonn.
`Thirty milliliters acetone was added to these
`extracts and the resulting solution titrated with
`0.1N acetous perchloric acid. Only intact
`procaine is measured in this technique. The
`percentage concentrations of the samples of
`procaine assayed were:
`Sample
`3
`
`l’eru-nlage Prnr-nine
`Control—— 1.91
`30 rniuutes—l.i)l
`60 minutes—l.!ll
`90 minutes—l.i)2
`1'20 miuutes—1.90
`180 minutcs—l.8‘J
`
`bdlGCJI‘.'1u$—
`
`MYLAN ET AL. - EXHIBIT 1020
`
`MYLAN ET AL. - EXHIBIT 1020
`
`

`
`l\"ll:rnt1:r"‘
`
`HEAT STERILIZATION AND LOCAL ANESTHETICS
`
`373
`
`The conclusion of the assay was: “Since one
`of the sarnples presumably represents a non-
`autoclazcd control, it follows that the slightly
`low as...1y values have nothing to do with the
`autoclaving;
`that
`is,
`there is no evidence of
`decomposition.”
`Tclrmrainc. Since there was a large number
`of tetracaine samples, selection for assay \vas
`made as follows: melting points were taken
`of the contents of each ampule. Those having
`a high melting point
`(145°—150° C.) were
`put aside as being of unquestioned excellence.
`Those with lower melting points were sub-
`jected
`to the more meaningful assay for
`p-hutylaminobenzoic acid (BABA),
`the hy-
`drolysis product of
`tetracaiue. Two of
`the
`20mg. size ampules and 5 of the 250-mg.
`size ampules were thus examined. The results
`are shown in table 1. These melting points
`demonstrate an interesting characteristic of
`tetracaine hydrochloride. This drug exists in
`three polymorphic crystalline fonns melting,
`respectively, at about 134° C., 130° C., and
`149° C.
`It generally consists of the highest
`melting form. Obviously, at autoclaved tem-
`peratures some conversion to the lower melting
`fonns occurs. However, further check was
`made for BABA. The values indicate negli-
`gible decomposition,
`if any, produced by
`nutoclaving.
`(2 per cent). Five-milliliter
`rllepiuacaine
`samples were made alkaline with 5 ml. of 10
`per cent sodium chloride and the solution ex-
`tracted 4 times with IS ml. of methylene
`
`'l‘.uu.r-1 1.
`
`Control
`30 minutes
`60 minutes
`90 minutes
`120 minutes
`180 minutes
`
`Percentage
`A mutant.MBA
`
`Results of Assays of 't'etrru-nine
`after Autoclaviug
`Melting: Point.
`(degrees ccntigmdc)
`’1‘etrncnine 20 mg.
`1-t5.6—1-16.8
`1-18.8-150.8
`145.2-140.0
`1303-13-1.8
`1-t6.0—l48.—t
`137.3-139.0
`
`0.07
`0.3
`
`Tetracn
`
`Control
`30 minutes
`(30 minutes
`90 minutes
`120 minutes
`180 minutes
`Pure drug
`
`inc 250 mg.
`121.8-133.2
`130.8—13-1.2
`135.~1—1~t2.2
`129.-1-135.4
`1~tti.8—l-19.2
`12-t.8—13-1.0
`1-15.0—150.0
`
`0.01
`0.02
`0.02
`0.02
`0.01
`0.02
`0.02
`
`’1‘.un.r-: 2.
`
`Results of Assays of .\Iepivacuinc
`After Autocluving
`
`S
`xiilliiill
`1
`2
`3
`4
`5
`6
`
`'
`.\ut1;'c'il.°»-ca
`180 minutes
`Control
`00 minutes
`00 minutes
`30 minutes
`120 minutes
`
`Vetting Point. of
`Percentage
`-I.
`.
`C°':§°{‘;{_fg‘°“ MI
`1.95
`In cacti case, the
`1.88
`melting point
`1.02
`of the e.\’tI'uclc(l
`1.91
`mepivncaine
`1.9!)
`base was be-
`2.00
`tween: 150°-
`153° C.
`
`TAHLE 3. Results of Second Study of Stul)ilit.y of Mepivncairie During Autocluving
`Melting Point of Extracted
`.\1cpi\'acnine Duo
`(degrees centigrulle)
`149.6—l5l.(i
`149.1-—l51.3
`t-1113-151 .0
`1~1t1.3—150.£)
`149.3-151.2
`1-t9.3—151.3
`
`1\|gnOl(l3l1I1:l\‘;Ed
`1
`2
`3
`«1
`5
`0
`1
`2
`3
`f.‘ 4
`5
`6
`
`\\'eig(lE§':7|t)Bnse
`0.0882
`0.0878
`0.0873
`0.0870
`0.0873
`0.0871
`0.0801
`0.0371
`0.0870
`0.0332
`0.0874
`0.0878
`
`file]
`
`0.1014
`0.1010
`0.1003
`1.1000
`1.1003
`1.1001
`0.0993
`0.1001
`1.1000
`0.1014
`0.1004
`1.1010
`
`Concelrtgpggiggct Drug
`2.03
`2.02
`2.01
`2.00
`2.01
`2.00
`1.08
`2.00
`2.00
`2.03
`2.01
`2.02
`
`11
`
`.
`1.
`
`tl1
`
`Exuiouucob
`
`

`
`374
`
`L. D. BRIDENBAUCH AND D. c. MOORE
`
`A...ai...aa..,,
`-‘[3!’-June 1964
`
`’1‘.un.r: «I. Results of Assays of Chlnroprorainc Mn-r Autoclaving
`
`Sm-mu.
`
`I
`2
`3
`-1
`5
`(i
`
`'”""‘.
`"'”"“'"""'
`
`1'20 minutes
`Control
`110 minutes
`180 minutes
`30 minutes
`60 minutes
`
`um.-1.
`
`(i00!)—00-I3
`6009-0l)«l3
`0009-0043
`(i0l2—05—
`
`6012-0525
`
`Wllalllilll-"'i'hl.)'l.T"
`prnrninn HCI
`
`""'“""“'=.“
`"3"""'>'-W
`
`3.58
`1.88
`1.88
`1.1!!)
`— — — Broken — — —
`L00
`-1.68
`1.97
`3,09
`1.95
`3.135
`
`These were pooled, dried with
`chloride.
`sodium sulfate, the sodium sulfate washed with
`several small portions of solvent. All solvent
`portions were then pooled and evaporated.
`The residues were weighed and the melting
`points taken (table 2). The values for samples
`2, 3, and 4 were slightly low, but
`it was
`thought unlikely that this indicated decompo-
`sition. More likely some analytical variation
`was involved.
`In order to verify this sup-
`position that only analytical variations were
`involved, additional assay was thought desir-
`able.
`In another test of stability twenty-four
`50-ml. commercial vials of 2 per cent mepiva-
`caine
`hydrochloride
`solution were
`used.
`Twelve were reserved as controls and twelve
`were autoclaved at 260° F.
`for 180 minutes
`at 20 pounds pressure. The autoclaved sam-
`ples were slightly discolored.
`Six of the con-
`trol vials and six of the autoclaved vials were
`assayed as before (table 3). These results
`apparently confirm the impression that mepiva-
`caine was not appreciably altered by heat
`sterilization and that minor variations in con-
`centration were due to analytical variation.
`Chloroprocaine. The autoclaved vials of 2
`per cent chloroprocaine were sent to the manu-
`
`TABLE 5. Assays of Samples of Same Lot. of
`Chloroprocainc After Autoclnvlng
`
`l
`l’
`.
`{‘;;;°,$:,':;3,§,g
`c...§§.E".2.l.‘:.“.’.’..e
`A,,3;;-;;,°,.,,,
`s..n...x..
`3.28
`1.95
`90 minutes
`1
`3.1 I
`1.98
`30 minutes
`2
`3-95
`1.98
`120 minutes
`3
`3.83
`1.95
`60 minutes
`-1
`1.10
`1.06
`Control
`5
`
`
`
`180 minutes 1.906 4.44
`
`facturer for assay. Sample 3 was broken in
`transit. The results of the analyses were re-
`ported as shown in table 4. REPORT: “It ap-
`pears that
`this experiment was set up with
`materials from two different lots.
`It would be
`desirable, if possible,
`to set up an experiment
`of this type with materials from the same lot.
`Label
`identifications were dilficult
`to read
`because of the breakage of a sample in transit."
`Because of the request for assay of samples all
`from the same lot six more bottles of 2 per cent
`chloroprocaine were autoclaved in the same
`manner. The assay was repeated and the
`results obtained as shown in table 5. REPom':
`"Since all of these vials came from our but
`1421,
`the initial assays on this lot were:
`percentage chloroprocaine HCl:1.97. Ap-
`parently there was no significant loss of potency
`from the repeated sterilization."
`Lidocaine (2 per cent).
`Lidocaine was
`subjected to both chemical and biological assay
`by the manufacturer (table 6). These results
`further support
`the established findings that
`lidocaine solutions, without epinephrine, may
`be reautoclaved without deterioration in bio-
`logical potency. The difference in the duration
`of motor block for the two sites of injection
`is due to the fact that the degree of vascularity
`of the two sites is different.
`Hexylcaine (2 per cent). The samples were
`assayed by the manufacturers for intact he.\'yl-
`caine
`hydrochloride
`and
`total
`hexylcaine
`hydrochloride (table 7).
`Piperominc (1.5 per cent 200 ml. vial)
`fonvarded to the manufacturer and analyzed
`for potency (method not
`reported).
`The
`results are shown in table 8. Since the theo-
`retical value is 15 mg./ml. (1.5 per cent), no
`significant decomposition is indicated during
`autoclaving of these samples.
`
`

`
`A |Im
`vVoIun£r“
`
`HEAT STERILIZATION AND LOCAL ANESTHETICS
`
`375
`
`'l‘Anl.l-I 6. Clu-micnl and llinlugit-nl .-\.‘i§l|_\‘S of l.i(lo('nin(- After Autm-lavin|.:
`
`~'.«-rilizinfl yiinw
`
`Siunnln
`
`l,"'l‘:'§“'l‘:)‘;fi"nlelCl
`
`1:11 Value
`
`(,'IwmiruI .-1ssa_:/: (Tests performed at room temperature of 73° 1".)
`
`Control
`:10 minutes
`60 minutes
`90 minutes
`I20 minutes
`1S0 minutes
`
`-1
`5
`1
`6
`3
`2
`
`1.98
`1.98
`1.98
`1.98
`1.98
`1.98
`
`6.62
`6.60
`6.62
`6.61
`6.60
`6.61
`
`I
`
`we
`
`.
`-
`-
`S‘°T':£:::"“
`
`
`
`cw
`
`
`
`-
`.
`'"e.;,ts"‘
`
`I
`
`"‘“ ‘““‘
`
`
`
`“““°''--i-
`
`
`
`"'°;'.a.;‘.'t?‘°'
`
`A '. D
`1'
`‘ J.‘.'.?§i;
`
`
`
`BiaIo_I/iml Asstty (wliilc rut srialir nerve injcrlion)
`
`-1
`5
`I
`6
`3
`‘2
`
`4
`5
`1
`6
`3
`2
`
`Control
`30 minutes
`60 minutes
`90 minutes
`120 minutes
`180 minutes
`
`Control
`30 minutes
`60 minutes
`90 minutes
`120 minutes
`180 minutes
`
`.
`
`I)
`E
`A
`F
`C
`B
`
`1)
`E
`A
`F
`C
`B
`
`0.1
`0.1
`0.1
`0.1
`0.1
`0.1
`
`0.2
`0.2
`0.2
`0.2
`0.2
`0.2
`
`R
`R
`R
`R
`R
`R
`
`L
`L
`L
`L
`L
`L
`
`Hip
`Hip
`Hip
`Hip
`Hip
`Hip
`
`.\1irlthig11
`.\1idthigh
`Midtliigli
`Mitlthigh
`.\li(lthigh
`Mitlthiglt
`
`12/12
`12/1‘.’.
`12/12
`12/12
`12/12
`1'2/12
`
`6/6
`5/6
`5/6
`5/6
`6/6
`6/6
`
`10-1
`8-!
`109
`101
`102
`I04
`
`103
`88
`85
`84
`90
`94
`
`Dilmcm'ne (121500, 20-ml. ampulc, each ml.
`containing 0.667 mg.) samples were sent
`to
`the manufacturer
`(method of assay not re-
`ported). The results are shown in table 9.
`1t is important to note that dibucaine in heavy
`solution should be resterilized only once be-
`cause further sterilization results in a darken-
`ing of the solution due to carmelization of
`the dextrose.
`
`Discussion
`the la\v suit of
`With the publication of
`the Ministry of
`Wooley and Roe versus
`Health,'~'-’ in which the legal decision stated
`that it was necessary to heat sterilize ampules
`of local anesthetics used for spinal anesthesia,
`the autoclaving of local anesthetics became
`establislied. However,
`there appeared to be
`no uniform method—some autoclaving the
`
`TABLE 7. Results of Assays of Hexylenine
`After Autoelaving
`
`TAIILE 8. Results of Assays of Piperocaino
`After Autoclaving
`
`-1
`6
`5
`2
`1
`3
`
`Control
`30 minutes
`60 minutes
`90 minutes
`120 minutes
`180 minutes
`
`19.6
`18.3"
`19.7
`17.8
`19.9
`16.8
`19.6
`17.5
`19.6
`17.1
`— — — Broken — — —
`
`1 (Lot 703119)
`2 (Lot. 703119)
`3 (Lot. 703119)
`4 (Lot 703119)
`5 (Lot. 703119)
`6 (Lot 727715)
`
`Control
`180 minutes
`90 minutes
`60 minutes
`120 minutes
`30 minutes
`
`1-1.7
`14.7
`1-1.7
`1-1.8
`14.7
`14.5
`
`3:17
`3.50
`3.40
`3.50
`3.39
`3.37
`
`

`
`376
`
`L. D. BRIDENBAUCH AND D. C. MOORE
`
`Anatliesiol
`.\lay—June lggty
`
`'I‘Am.l-: 9. Results of Assays of Dibucaino
`After Aulm-lnving
`
`Ampule
`1
`2
`3
`4
`5
`6
`
`Time .-\IItncln\'Nl
`90 minutes
`I20 minutes
`Control
`60 minutes
`180 minutes
`30 minutes
`
`0.65‘
`0.65‘
`0.65’
`0.65‘
`0.6-!‘
`0.61‘
`
`ampules in which the lumbar puncture sets,’
`some autoclaving only the ampules for spinal
`anesthesia, others advocating autoclaving all
`drugs and equipment used for regional anes-
`thesia.‘«‘v°
`Time and pressure techniques
`varied from 10 poundsfor 15 minutes to 20
`pounds for 45 minutes at the same tempera-
`ture.7
`In 1954, Whittet“ attempted to deter-
`mine the effects of multiple autoclaving of
`local anesthetic drugs for periods of from 1
`to 6 hours. There was deterioration within
`3 hours. The results of our study vary from
`Whittet’s study in that
`the tetracaine tested
`did not alter in potency after three hours of
`autoclaving. This may be due to the fact that
`both of the samples of tetraeaine tested con-
`sisted of niphanoid crystals, while that tested
`by Whittet was in solution, hence less stable.
`In the authors’ hospital, both heavy dibu-
`caine (l:200 in 6.0 per cent glucose) and
`heavy tetracaine (1 per cent in 10 per cent
`dextrose) are not autoclaved more than twice
`because of carmelization of the glucose and
`subsequent discoloration if heated longer than
`two hours.
`The results of the present study indicate
`that all of the commonly used local anesthetic
`drugs can be heat sterilized (18 pounds pres-
`sure, 260—275° F.
`for 30 minutes) at
`least
`6 times,
`for a total
`time of at
`least
`three
`hours without loss of potency. Cerlich et a1.“
`detennined that sterilization could be accom-
`plished at 15 pounds pressure and 250° F.
`temperature for 15 minutes. The system of
`heat sterilization using 18 pounds pressure at
`260-275“ F. for 30 minutes is recognized as
`being longer than necessary for sterilization
`of the drug. However, it was adopted because
`this is
`the routine for autoclaving surgical
`packs at our institution. Consequently,
`the
`regional block trays containing the local anes-
`thetic drugs could be included along with the
`
`surgical packs, thus decreasing the work neces.
`sary, and eliminating any special routine for
`sterilimtion. This system of autoclaving and
`reautoclaving of drugs has been in practice
`at our institution since 1953 and the drugs
`used in 30,979 cases without any case of in.
`fection or clinically observed loss of potency.
`
`Summary
`
`Heat sterilization of local anesthetic drugs
`has been established a necessity.
`In order
`to determine whether
`the newer anesthetic
`agents could be autoclaved without
`loss of
`potency and, if so, how many times, samples
`of all of the commonly used local anesthetic
`drugs were autoclaved for periods varying
`from 30 minutes to 3 hours.
`These were
`subsequently assayed by their manufacturers
`for potency. None of the agents tested showed
`any appreciable loss of potency following auto-
`claving at 18 pounds pressure, 260-275” F.
`for 3 hours.
`
`The authors wish to ex ress their gratitude to
`the research laboratories o Sterling-Winthrop In-
`stitutc, Strasenbur h Laboratories, Merck Sharp
`& Dohme Researci Laboratories, Astra Pl1am1a-
`ceutical Products,
`lnc., Eli Lilly and Company,
`and Ciba Phamiaceutical Company for their co-
`operation and aid in assa ing the dmg samples
`for potency following steri ization.
`References
`
`El!
`
`1.
`to
`
`.\ledico-Legal: Spinal anaesthetics appeals lail,
`Brit. Med. J. 1: 940, 1953.
`. Medico-Legal: Contaminated spinal anaesthetic,
`Brit. Med. J. 2: 1165, 1953.
`8. Walton, F. A.: Correspondence, A.\'E.S‘l'llF_§I-
`oLoc\' 13: 441, 1952.
`4. Bridenbaugh, L. 1)., and Moore, D. C.: Is heat
`sterilization of local anesthetic dmgs a ne-
`cessity?, ].A..\I.A. 168: 1334, 1958.
`Young, J. A., Collette, T. S., and Brehm, \V. F.:
`Sterility of multiple dose vials after repeated
`use, Amer. Surg. 24: 811, 1958.
`6. De Jong, R. H.:
`.\lultiple autoclaving of com-
`mercial
`local anesthetic solutions containing
`dilute epinephrine, ANes'r1n~:sio1.ocr 24: 582,
`1963.
`7. Carter, A. B., Herbert, C. L., DeWald, W. S.,
`and Talley, A. W.: Multiple autoclaving of
`drugs used in spinal anesthesia, ANes1'n£sr-
`or.ocr 15: 480, 1954.
`autoclaving in
`8. Whittet, T. D.: Effect of
`ampoulcs of local analgesics, Anaesthesia 9:
`271, 1954.
`9. Cerlich, N. A., Nicholas, P. S., and Ballingcr,
`C. M.: Heat sterilization of spinal anesthesia
`ampules (abstract), Am-;s11rEsroLocv 13: 164,
`1957.