Technical Notes
`
`Stability of Local Anesthetics After Autoclaving:
`Determined by Gas Chromatography
`Jordan Katz, M.D."
`
`TIIE I-JFI-‘l—3(Tl'S of autoclaving on the stability of
`four local anesthetics were analyzed using gas
`chromatography. One per cent solutions of
`the hydmchloride salts of pmmine (Novo-
`eain),
`lidocaine
`(Xylocaine), mcpi\acaine
`(Carbocaine), and prilocaine (Citanest) were
`steam-autoclaved fmm one to three times at
`250° C. for 20-30 minutes. The local anes-
`thetics rxrme from their original vials or nrbber
`stoppered ampules. Lidocaine hydrochloride
`contains 1 mg./ml. of metlrylparabcrr, and pro-
`caine hydmclrloride methylparaben 0.5 mg...’
`ml. and propylparabc-n 0.05 mg./ml.. as pre-
`sewatives.
`Stock solutions of prilocaine hy-
`drochloride and mepivae.rine hydrochloride do
`not contain preservatives.
`
`Method
`Five tenths milliliter of 2 .\' .\'aOH and 1.0
`ml. of carbon disullide (CS._.) were added to
`a 1.0 ml. sample of local anesthetic solution.
`The addition of .\'aOH liberates the local anes-
`thetic base from its hydrochloride salt and
`makes it soluble in the organic solvent.
`CS._. \vas chosen as the organic solvent since
`the flame detector is relatively insensitive to
`it and thereby separations \vere easily made.
`Thorough mixing was accomplished rising a
`Cyclo-mixer ' for 60 seconds. Gas chmmalo-
`graphic
`analyses were done
`in
`triplicate
`using an Aerograph Hi-Fi Model 600 Cl gas
`chmmatograph.
`
`' Assistant Professor. Department of Anesthesia.
`Stanford University School of .\lr.-dicine. Palo Alto.
`California.
`Accepted for publication May 17, 1966. Sup-
`ported in part by N.l.H. Grant GM 12527.
`' Clay-.-\dams Company. New York. New York.
`l Varian-Aerngraph Company. Walnut Creek,
`California.
`
`Canrliliam 0] Armlyi-r'.s': Column~stainless
`steel, 5 feet X ‘J. inch, 2 per cent SE 30 on
`100-120 mesh A/\V, Chrom \\’ D.\lCS; Col-
`umn temperature 190° 0; injection port tem-
`perature 250' C.; hydrogen flame detector
`(hydmgen flow = 30 ml./minute); Carrier
`gas—Helilun at 75 ml./minute 5
`injections
`\vere used.
`script writer
`and Northmpt
`A Leeds
`equipped with :1 Disc .\lodel 224 integrator
`was used to record the chromatogram.
`A
`typical tracing is shown in figure 1. The inte-
`grated area is a representation of the concen-
`tration of the drug. By comparing integrated
`areas between nonautoclaved controls and
`autoclaved samples. changes in concentration
`can be determined.
`
`Results
`
`Rtsults of the study are shown in table 1.
`The differences between autoclaved and con-
`trol gmups were not statistically significant as
`determined by the analysis of variance ' rising
`computer program B.\lD 06V (General Linear
`Hypothes'Ls with Contrasts).
`
`Discussion
`
`.\lethods utilized previoltsly for analyses of
`local anesthetics have involved dye complex-
`ing and quantitative colorimetric detennina-
`tions.‘-‘v‘ The complex occurs between the
`dye and the basic amine portion of the mole
`cule. Unless
`specific extraction techniques
`are used, other amines. metabolites or break-
`down products can not be distinguished. Fig-
`ure 2 shows a chromatogram of a single injec-
`
`tLee<ls and .\'ortlmrp Company, Philadelphia,
`Pennsylvania.
`81-?
`
`|\./IYLAN ET AL. - EXHIBIT 1021
`
`MYLAN ET AL. - EXHIBIT 1021
`
`

`
`JORDAN KATL
`'l‘.un.r: I.
`
`A ease.‘ I
`.\'o»7.—n.¢."'iWc
`Pen-1-ntagu Concentration of Autoeluved
`Dmgs Compaml with Controls‘
`
`.\'umber oi
`-rim"
`(\utodn\'«1
`l
`
`2
`
`.'I
`
`Lidnnine
`‘'5’
`
`-- ‘
`l0l.7
`-'.'.B
`+3.8
`~73
`+Z|.Z!
`-5.0
`
`iD0.I'n
`
`i|II.‘J
`
`.\l¢~;_-i-
`‘,n_um__
`(-7,)
`+1:
`90.5
`-4.1
`+6.1
`-1.-I
`+3.:
`-'.'.l
`
`D13.-'3
`
`1lI.'.'
`
`Pnucnine
`"70
`+1.15
`IDLO
`-9.1
`+8.8
`- Lli
`+12:
`- I213
`
`ll)'J.~'n
`
`HILL’
`
`Priloe-nine
`"3’
`-1-is
`97,!
`-' 1.1
`+3.]
`-IKJ
`+«;.-
`-L0
`
`l(l].D
`
`$18.3
`
`' Pen-onuxr Cunt-rritrntiun
`_ lnmmuni -Ira ul ...u.ei.m1 am "ll.
`lnmzm-i urn oi runlrol
`X
`
`tiou of the four local anesthetics used in this
`study, demonstrating that
`chromatographic
`separation, at
`these concentrations.
`is easily
`accomplished.
`Since this method can dis-
`tinguish between closely related basic amines.
`it should be able to detect any breakdown
`products caused by autoclaving. There was
`no evidence
`that destmctinn does occur.
`Other columns and conditions of an'al_\'sis have
`been used confirming the observation that the
`local anesthetic molecule appears stable to
`autoclaving.
`Gas chmmatographic analysis is applicable
`to clinical
`investigation.
`Several authors "‘
`have described methods which are extremely
`sensitive and reproducible at pliysiological
`concentration of the drug. A recent report“
`describes blood levels after intravenous injec-
`tions of lidocaine below 0.5 pg./ml. using gas
`chromatographic methods.
`
`Conclusions
`
`Commercially available ampules of the hy-
`drochloride salts of procaine, lidocaine, mepi-
`vacuine and prilocaine were exposed to stami-
`ard autoelaving conditions. Gas chromato-
`graphic analyses substantiated the observations
`that the concentration of local anesthetic is not
`altered by autoclaving. No breakdown prod-
`ucts could be detected.
`
`The author would like to express his appreci-
`ation to Gerald R. Chase, Ph.D. for help in ana-
`lyzing the data.
`
`Prllocolnz PZOK
`
`
`
`
`
`Fin. 1. Sample chromatogram of the deter-
`mination of prilocaine potency.
`
`_» mu... tc........)
`;
`s
`
`: ulozuu-4 t-._........)
`.
`:..,
`
`:-.,
`; »...~.-. ow.-co»-)
`.
`
`
`
`;~....m.... .cu»:.«..|
`
`:..,
`
`Fla. 2. Chromatogram of a mixture of four
`local anesthetics. Conditions of analysis: Same
`as in text, except
`that column temperature was
`lowered to 170° C.
`to aid in separation of the
`dmgs.
`
`

`
`Volume 17
`Number 6
`
`A.\'E$THETlC STABILITY AFTER AUTOCLAVINC
`
`837
`
`References
`1. Sehaife, H.: The Analysis of Variance. New
`York. John \Viley and Co., 1959.
`2. Brodie, B. B., Lief. P. A.. and Poet. IL: The
`fate of procaine in man following its intra-
`venous adlninistration and methods for the
`estimation of procaine and diethylaminoctha-
`nol, ]. Pharrnaeol. Exp. Ther. 94: 59. X948.
`3. Sung. C.. and Tmant, A. P.: The phy.\ioloy,:ir.-al
`disposition of lidocaine and its mmparison in
`sonic respects with procaine. J. Phanuaenl.
`Exp. Ther. I12: 43?. I954.
`
`-I. Svinhufvud, G., Ortengrcn, B., and Jaeobsson,
`S. E.: The estimation of lidocaine and prilo-
`caine in biologiml material by gas ehmma-
`tography, Scand. J. Clin. Lab.
`invest. 17:
`161 1965.
`5. Beckett, A. H.. Bayes, R. N., and Parker, J.
`B. R.: Determination of lignoeaine in blood
`and urine in human subjects undergoing local
`analgesic procedures, Anaesthesia 20: 29-1,
`I985.
`6. liargrove. R. L. Hoyle, J. IL, Parker, J. B. R..
`Beckett. A. IL, and Bayes, R. .\'.: Blood ligan-
`eaine levels
`following intravenous
`regional
`analgesia, Anarsthesia 21: 37,
`l96G.
`
`PERIDURAL COMPRESSION To investigate why the same level of spinal or
`epidural anesthesia is achieved with less mass of local anesthetic in late pregnancies
`than in the nonpregnant state, radiogmphic measurements were made of the effect
`of inferior vena eaval occlusion on the spread of epidural and intmthecal contrast
`in dogs. The increase in pressure in the inferior vena eava caused by
`material
`inflation of a culled catheter resulted in marked cephalad movement of epidural con-
`trast material. A Valsalva maneu\'L'r prrxluced the same results. Neither inferior
`vena caval obstmetion nor Valsalva maneuvers caused comparable movement of in-
`tratheeal contrast material.
`(llipona. F. A., Tales, 11.. and Ila-lire, F. \V.: Venom
`Eneroaclrmen! an the Spinal Peridurul Space Due to Experimental NC Occlusion,
`lm:e.\1. Radial. 1: 157 (March) 1966.)
`
`RENAL HEMODYNAMICS The combined eilects of unilateral renal vasodiIata-
`tion and angiotensin infusion on renal hemodynamics and sodium excretion and
`realisorption were studied in anesthetized hydropcnie dogs. Unilateral renal vaso-
`dilatation alone with either acetyleholine, bradykinin. or kallidin resulted in an
`ipsilateral increase in renal plasma flow and an ipsilateral decrease in net tubular
`lnfusion of angiotensin or norepinephrine in the presence
`rmbsorption of sodium.
`of unilateral renal vasodilatation resulted in a sustained marked increase in sodium
`excretion and decreased sodium reabsorption by the \".Lsodilatcd kidney. These
`changes occurred in association with decreases in glomerular filtration rate. clearance
`of PAH. renal plasma flow. and nonwrtical plasma flow. Sodium excretion usually
`decreased in control nonvasodilatcd kidneys during the infusion of angiotensin or
`norepinephrine, although glomerulnr filtration rate was often similar in the two
`The cleamm.-e of PAH. however, was always distinctly lower in the can-
`kidneys.
`trol nonvasodilated kidney.
`The results are consistent \\1'th the \’iC\\' that proper
`combination of two ph_\'siologirral|_\' important variables, arterial pressure and renal
`vascular resistance, can effect large changes in the tubular reabsorption of sodium.
`Changes in these two variables may be
`probably through intmrenal mechanisms.
`(Sarlei. L. E.. and
`of major importance in the regulation of sodium excretion.
`Friedler, R.
`.\l.: Eflects ni Combined Renal Varodilutation and Presser Agents on
`Renal llcmodynamics and the Tulmlar Heal»-orption of Sodium. J. Clin. Incest. -I5:
`542 (April) 1966.)