High-Performance Liquid Chromatographic Method for the
`Determination of Esmolol Hydrochloride
`
`YINC-CHI LEE, DAVID MICHAEL BAASKEX, and ABU S. ALAM
`Rcceived September 14. 1983. from the Pharmaceurical Development Department. Research and Decelopment Dicision. American Critiral Care, McGaw
`Accepted for publication January 13, 1984.
`Park, IL.60085.
`
`.
`
`ocn,cn-cn,
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`cicn,cn- / O \ cn,
`
`Abstract 0 A rapid high-pcrformancc liquid chromatographic method for
`the determination of esmolol hydrochloride. a ncw ultra-short-acting beta
`blocker. is described. The stability-indicating nature of the method was
`demonstrated by resolving esmolol from synthetic intermediates, potential
`impurities, and the product ofdecomposition. Reverse-phase liquid chroma-
`tography was performed with a microparticulate ( 10-wn) cyano-bonded
`silica-packed column, a fixed-wavelength UV absorbanccdctector (A = 280
`nm), and a mobile phase of acetonitrile-0.005 M sodium acctate-acetic acid
`( I 5:84: I ) pumped at 2 mL/min. The internal standard was 2-p-chlorophe-
`nyl-2-methylpropanol. A percent RSD of < I .7% and an accuracy ( IOWO -
`mean error) of >98.6% were achieved over the concentration range studied
`(100-500 pg/mL). with corrclation coefficients >0.9996.
`Keyphrases 0 HPLC--determination of esmolol hydrochloride 0 Esmolol
`hydrochloride-- HPLC
`
`Esmolol hydrochloride (methyl 3 [ 4-[2-hydroxy-3-[( 2-
`met hylet hy1)aminoj propoxy] phenyl] propionate hydrochloride;
`I ) is the first of a new class of beta blockers, known as ultra-
`short-acting beta blockers, to enter clinical trials. The ultra-
`short-acting beta blockers were designed to extend the use-
`fulness, safety, and efficacy of beta blockers in critical cardiac
`therapy through controlled and titratable intravenous therapy
`( 1 ). This was accomplished by designing chemical instability
`into the molecule. The ester functionality of the molecule is
`susceptible to cleavage by the nonspecific serum esterases to
`the free acid 3 [ 4- [ 2- h ydrox y-3- [ (2-methy let hy1)am in01 -
`propoxy ] phenyllpropionic acid.
`Esmolol hydrochloride was synthesized (2) from 3-@-
`hydroxypheny1)propionic acid (I I) uia a four-step process
`(Scheme I). In this report the development and validation of
`a high-performance liquid chromatographic (HPLC) method
`for the quantitation of esmolol in the presence of synthetic
`intermediates (11-IV) and free acid (V) is described.
`
`EXPERIMENTAL SECTION
`Materials--Compounds I, Ill, IV. V.and VI were synthesizcd in this lab-
`oratory. 2-@-Chloro-phenyl)-2-methyl propanol’, compound 11, glacial acetic
`acid*. and sodium acetate2 were used as received. Class-distilled acetonitrile
`and methanol were used for all proccdures3. Purified water4 was used
`throughout.
`liquid chromatographic system (3). equipped with
`Chromatography-A
`a fixcd-wavelength UV absorbance detectorS at 280 nm. an on-linc data sys-
`tern6, and a column (30 cm X 3.9 mm) packed with cyano-bonded silica
`( IO-pm)’. was used. Sample injections were 50 pL. Mobile phase was prepared
`fresh daily by thoroughly mixing 150 tnL of acetonitrile, 10 mL of glacial
`acetic acid. and 840 mL of sodium acctate trihydrate (0.068%. w/v) buffer
`which WBS filtered through a 0.5-pm filters prior to use. h constant flow rate
`of 2 mL/min yielded a pressure of <2000 psi.
`
`I CH,CH,CO,R’
`
`V
`
`&H,CH,CO,R’
`
`I
`
`R ’ = H : R ’ = CHI
`Scheme I
`
`Standard concentrations of 500,400. 300,200. 100, and 0 &mL were
`prepared in quadruplicate. To I .O ml. of each standard or sample was added
`750 pL of the internal standard 2-(p-chlorophcnyl)-2-methyl propanol (4
`mg/mL) in methanol-water (5050). Peak arcas were measured. and the ratios
`for esmolol-internal standard were calculated with the data system, yielding
`a calibration curve. Samples were prepared in water at 500pg/mL.
`Spedficity of the Metbod-Esmolol hydrochloride (50 mg) was placed in
`each of four 100-mL volumetric flasks. Into each flask was added 75 mL of
`either water 1 M HCI, I M NaOH, or 3090 hydrogen peroxide. The flasks were
`gently boiled for I h. After cooling. the pH was adjusted to 4 with concentrated
`
`I
`
`I Aldrich Chemical Co.. Milwaukce. Wis.
`Analytical reagent; Mallinckrcdt. St. Louis. Mo.
`J Burdick & Jackson I.aboratories. Muskegon. Mlch.. or J. T Baker Chemlcal Co..
`Phillipsburg. N.J.
`MiIIi.Q Watcr Purification System; Millipore Corp , Bedford, Mass.
`5 Model LC- 15. Perkin-Elmer Corp.. Norwalk. Conn.. or model 440; Waters Asso-
`c i a ~ o . Milford. Mass.
`’ p-Bondapak CN; Waters Associates.
`tlP.3354: Hewlett-Packard, Avondalc. Pa.
`Uillipure Corp.
`
`m
`
`
`3
`0
`9 12
`Figure I - - Typical chromatogram showing the resvlurion (111 from the syn-
`(hetic intermediates (II-IV) and rhe anricipoted breakdown produel (VI. I S .
`internal standard.
`
`1660 I Journal of Pharmaceutical Sciences
`Vol. 73, No. 11. November 1984
`
`0022-3549184/ 1 100- 1660SO 1.0010
`@ 1984, American Pharmaceutical Associatlon
`
`MYLAN ET AL. - EXHIBIT 1007
`
`

`

`I
`
`V
`
`V
`
`I.S.
`
`I.S.
`
`I.S.
`
`'t.
`
`I;.
`
`rn m rn n - I
`
`0
`1 0 0 5 1 0 0 5 10
`1 0 0 5
`5
`Figure 2-Chromatogram of esmolol hydrochloride solurions boiled for I
`h. Key: (A) water. pH 5.5; (B) I M NaOH; (C) I M HCI; (Dl 30% HzO2.
`
`HCI or 10 M NaOH. All flasks were brought to volume with water and ana-
`lyzed.
`
`RESULTS AND DISCUSSION
`
`The direct measurement of the raw drug for esmolol hydrochloride content
`in the presence of the anticipated synthetic intermediates, the synthetic starting
`material, and the anticipated breakdown product is shown in Fig. 1. The de-
`tector wavelength (280 nm) was chosen to enhance visualization of all potential
`
`Table I-Analysis of Four Experimental Lots of Esmolol Hydrochloride
`
`Lot
`A
`B
`C
`D
`
`Mean,
`%
`97.8
`91.4
`98.6
`97.5
`
`RSD,
`%
`1.15
`1.23
`I .95
`0.8 I
`
`Number of
`Determinations
`24
`24
`21
`18
`
`Time
`2 years
`2 years
`I year
`9 months
`
`synthetic intermediates and not for maximum sensitivity for esmolol. The limit
`of quantitation for esmolol hydrochloride was -I0 pg/mL under the reported
`operating conditions.
`Applicability-The specificity of the HPLC system was tested with de-
`graded esmolol samples. No changes in I concentration were seen in the boiled
`aqueous solution. After 1 h in boiling acid ( I M HCI) or base ( I M NaOH),
`I was almost completely converted to V, as might be expected under these
`conditions. Finally, boiling for I h in 33% hydrogen peroxide yielded several
`additional unidentified products (Fig. 2). In each chromatogtam, it can be
`seen that the size of the I peak decreases with degradation. The practicality
`of the method was demonstrated by the analysis of four synthetic lots (Table
`I). The percent RSD values for the analysis over a 2-year period and with
`several analysts were consistently <2%.
`Accuracy and Precision-ln
`spite of consistently high correlation coeffi-
`cients (>0.996), the peak height ratio method was not employed, as erroneous
`results were obtained due to tailing at higher concentrations. Curvature or
`tailing did not influence the peak area ratio calculations which were employed
`for all studies. Data generated by three separate analysts on each of 3 d yielded
`an accuracy >98.6%, percent RSD of < I .64%, and correlation coefficients
`>0.9996 for thecalibration curves. The percent RSD valucs for a single sample
`were <2% (Table I).
`
`REFERENCES
`( I ) J. Zaroslinski, R. J. Borgman, J. P. ODonnell. W. G. Anderson. P.
`W. Erhardt, S.-T. Kam, R. D. Reynolds. R. J. I.ee. and R. J. Gorc7ynski. Lije
`Sci., 31,899 (1982).
`(2) P. W. Erhardt, C. M. Woo, W. G. Anderson, and R. J. Gorczynski,
`J. Med. Chem., 25,1408 (1982).
`(3) D. M. Baaske, N. N. Karnatz, and J. E. Carte;, J. Phurm. Sci., 72, 194
`(1983).
`
`ACKNOW LEDCMENTS
`
`The authors thank Dr. Paul W. Erhardt and Mr. Chi M. Woo for the syn-
`thetic products cited and Ms. Lori Coomans for assistance in the preparation
`of this manuscript.
`
`Effect of Ethanol, Glycerol, and Propylene
`Glycol on the Stability of Phenobarbital Sodium
`
`V. DAS CUF'TA
`Received October 18, 1983, from the Department of P'harmaceutics. Unioersity of Houston, Houston. TX 77030.
`19, 1984.
`
`Accepted for publication January
`
`Abstract 0 The effects of ethanol, glycerol, propyleneglycol, phosphate buffer,
`and ionic strength on the stability of phenobarbital sodium have been studied.
`Ethanol had the maximum stabilization effect followed by propylene glycol
`and glycerol when compared with the stability in water. The estimated half-
`
`lives at 5OoC (pH - 8) were 78.95. 109, and 127 d in water and 2w0 aqueous
`
`solutions of glycerol, propylene glycol, and ethanol. respectively. The effects
`of phosphate buffer and ionic strength were negligible.
`Keyphrases 0 Phenobarbital sodium-stability, cffectsof ethanol, glycerol,
`and propylene glycol 0 Stability-phenobarbital, effect of solvents
`
`It is well known that thc stability of phenobarbital in liquid
`dosage forms depends on thc pH and the vehicle. A common
`method to minimize dcgradation (1) is to use a mixed solvent
`of water and an organic solvent such as ethanol, glycerol, or
`propylene glycol. The stabilization effect of ethanol is thought
`to be due to a decreased dielectric constant (2), which slows
`
`down the reaction between ions of like charges, i.e., the ionized
`form of phenobarbital and the hydroxyl ions.
`An earlier report (1) indicated that it was difficult to select
`a stability-indicating method for the quantitation of pheno-
`barbital. Recently, a stability-indicating assay method (3)
`based on HPLC has been rcported which is applicable to liquid
`
`0022-3549/84/ 7100-1661$0 1.0010
`@ 1984, American Pharmaceutical Association
`
`Journal of Pharmaceutical Sciences I 1661
`Vol. 73, No. 1 1 , November 1984
`
`