1587
Anal. Chem. lQ86, 58, 1587-1589
C
A
4 I Flgure 4. Typical flow lnjectlon signal profiles for inlection of penicillln G Into a 1 mM phosphate buffer carrier of pH 6.40 and 0.10 M ionic strength: flow rate, 4.0 mLlmin; injected sample size, 70 KL; temperature, 25 OC. Millimolar concentrations of penlcillin G injected Indicated under each set of peaks: (a) 3.6-m borosilicate glass (ammonium hydrogen fluoride etched) open tubular reactor (0.8 mm i-d.); (b) 0.35-m CPG-Tygon reactor (1.0-mm4.d. Tygon tubing), CPG, 80/120 mesh, 75-A nomlnal pore dlameter; (c) 0.55-m CPGTygon reactor (1.Omm-i.d. Tygon tubing), CPG, 200/400 mesh, 3000-A nomlnal pore dlameter.
times as good for the reactor with CPG of 75-A pore diameter. The reactor length was roughly l/lo as large with the CPGTygon reactors, and normalization of peak height/reactor length shows the CPG-Tygon reactors to be 15-18 times as good as the borosilicate glass reactors. As stated earlier, the same reactors are about 37 times better than borosilicate OTRs when glutaraldehyde immobilization is considered. The difference results from the fact that the enzyme does not
utilize all reactive aldehyde groups available. Independent measurements (10) have shown, for instance, that for an average 46 f 15.8 nmol of aldehyde groups, only an average of 7.8 f 2.6 nmol of immobilized protein is measured in the same borosilicate OTR. The comparative performance studies reported above clearly indicate the advantages of using the novel design for immobilized-enzymereactors in continuous-flowsystems. The better uniformity and higher local activity of the reactors are of considerable analytical interest; their simplicity and fast preparation, however, should be of perhaps even greater appeal to those interested in making immobilized-enzyme reactors for continuous-flow systems. A CPG-embedded-on-plastic reactor can be made in a few minutes, whereas the elaborate procedural steps of whisker growth (1-3) may very well take as much as 3 days and involve the use of corrosive chemicals such as refluxing concentrated HCl and etching with NHdHFz a t 450 "C.
ACKNOWLEDGMENT The help of Pamela Schwager and CONOCO, Inc. (Ponca City, OK), with the scanning electron micrographs is gratefully acknowledged here. Registry No. Penicillinase, 9001-74-5. LITERATURE CITED (1) Iob, A,; Mottola, H. A. Anal. Chern. 1980, 52, 2332-2336. (2) Iob, A.; Mottola, H. A. Clln. Chern. (Wlnsfon-Salem, N.C.) 1981, 2 7 , 195. (3) Gnanasekaran, R.;Mottola, H. A. Anal. Chern. 1985, 57, 1005-1009. (4) Mottola, H. A. Anal. Chlm. Acta 1983, 145, 27-39. (.5.) Pierce 1985- 1986 Handbook and General Catalog; Pierce Chemical Co.: Rockford, IL, 1985; p 186. (6) Gosneii, M. C.; Mottoia, H. A. Anal. Chem. 1986, 58, 631-636. (7) Modern flasfics Encydopedia ; McGraw-Hill: New York, 1984165; Vol. 61, NO. 10, pp 24-26. (8) Bulletin, T-104, Norton Industrial Plastics, Akron, OH. (9) Anderson, L. D., Norton Industrial Plastics, personal communication. (10) Gosnell, M. C., Oklahoma State University, unpublished results, 1986.
RECEIVED for review December 5, 1985. Accepted January 27, 1986. This research has been supported by the National Science Foundation (Grant CHE-8312494).
Stirring Device for Direct Microtitratlon of Pilocarpine Hydrochloride in Individual Single-Dose Containers Per-Arne Johansson Analytical Control, Astra Pharmaceutical Production AB, S-151 85 Sodertalje, Sweden Content uniformity is determined routinely for many solid formulations in the quality control of drugs (1). The total drug content of a liquid formulation in an ampule, cartridge, pipet, etc., may also be of interest, e.g., in connection with validation or trouble-shooting of a manufacturing process. Instead of transferring a liquid sample to a volumetric flask and running a spectrophotometric or chromatographic assay, it should be possible to determine the drug content by titrating directly into the drug container. If the drug container is very small, this can only be accomplished after miniaturization of the titration. Microscale acid-base titrations have been used for many years in the pharmaceutical analysis, but mainly for qualitative purposes. A typical example is the pK, determination using a milligram sample in a preformulation study (2).
Stirring in microscale titrations can be achieved in various ways, e.g., by magnetic stirring (3),by swirling of the titration vessel (41, by spinning the sample (5,6), or by blowing nitrogen or air through the sample solution (2, 6). All these stirring devices are based on specially designed titration vessels into which the sample solution must be transferred. They are therefore less suitable for in situ titrations of drugs in small-volume drug containers. In the present work, a stirring device for microscale titrations has been developed in which the drug container is used as a titration vessel. The stirrer was constructed from commercially available plastic building components (Technic Lego) and was tested by determining the acidity constant of the pilocarpinium ion and the content of pilocarpine hydrochloride in individual single-dose eye-drop pipets.
0003-2700/86/0358-1587$01.50/00 1986 American Chemlcai Society
1688
ANALYTICAL CHEMISTRY. VOL. 58. NO. 7. JUNE 1988
M2.
Shdedme ppet (A) fat tab. (B) mntaher W .(C) bottom.
3.75 mV/min and a medium measuring-point density; cf., ref 7
-. z.
Fipn 1. TlbatJon aassmbiy.
and 8. Acidity Constant Determination. The acidity constant of the piloearpinium ion was obtained from the titrations above in two ways: by calculations according to Albert and Serjeant (2) using the measuring-point list of the titration (multiplepoint evaluation) and by taking the pH value at 50% neutralization (single-point evaluation). The latter value is printed out after each titration if the automatic titrator has been programmed via the control card and this bas been marked in the pK. field (9). The pK. value determined in this work is a "mixed" constant and incorporates hydrogen ion activity and molar concentrations of the acid-base pair. The determinations were carried out in room temperature (about 22 "C) at an ionic strength of about 0.1.
-
EXPERIMENTAL SECTION Apparatus. The design of the titration assembly is shown in Figure 1. The stirring device WBB constructed hy using a Technic Leg04.5V motor (no. 8700, Leg0 System A/S, Billund, Denmark), a gear block (no. 872, Leg0 System A/S), two supplementary sets (no. 8710, Leg0 System A/% and a combination pH microelectrode (no. 10 402 3167, Ingold AG, Urdorf, Switzerland). The motor was driven by a dc power supply (type 719, Elfa. Solna, of a O.Frmm-i.d. x 1-mm-0.d.Teflon tubing Sweden). Two pidrawn into a 0.8-mm-0.d. capillary were amched to the electrode stem by three 1-mm Tygon sleeves that were cut from Tygon pump tubes (Technicon Carp.,Tarrytown, NY). The titrant was delivered through one of the Teflon capillaries that ended about 3-4 mm above the junction. The other capillary served as a 'stirrer" and ended 9 1 0 mm below the electrode tip in order to reach the bottom of the narrow, conical veasel that ia formed when the pipet is in the upside-down position; cf., Figure 2. Mixing of sample and titrant was achieved by revolving the titration veasel (in this ease the single-dose pipet) while the electrode with attached tubing remained stationary. An automatic titrator (Titroprocessor 636 with Program 102, Metrohm AG, Herieau, Switzerland)with a 1-mL buret wan used in the titrationa Titrant volumes of 1pL can be delivered from the buret during a titration (7). Reagents and Chemicals. The titrant, 0.1 M NaOH, was prepared from sodium hydroxide ampules (Titrisol, Merck AG) using carbon-dioxidefree water. Piloearpine hydrochloride and solutions of piloearpine hydrochloride (pilocarpineeye drops 2% single-dose pipets, Tika AB, Lund, Sweden) were of pharmacop i a l grade. Titration Procedure. Place the pipet (Figure 2) on the 8 t h gem wheel with the flat tab (A) downward and the container M y (B)inside the four des; see Figure 1. Cut off the bottom of the pipet at C using a scalpel. Add 5.0 r L of 1 M HCI by use of a pipetter (Micro/Pettor, Model C, SMI, Inc., Emeryville, CA). Titrate with 0.1 M NaOH under a stream of nitrogen (cf.,Figure 1) using a stirring speed of about 200 rpm. Run the automatic titrator in the dynamic titration mode, using a drift criterion of
~
RESULTS AND DISCUSSION Titration of Pilocarpine Hydrochloride. The pilocarpine cation is a weak acid with a pK. of about 6.9 (10). However, titration error calculations (see,e.g., ref 11) indicate. that it should be possible to titrate such weak acids in aqueous solutions (with NaOH) with an error of 0.1% or leas. The present eye-drop preparation WBS formulated amrding to Gibbs and Tuckerman (12) and is practically unbuffered only a small amount of NaOH is added to an aqueous solution of pilocarpine hydrochloride in order to raise the pH to about 5.3. Since both piloearpine hydrochloride and piloearpine base are present in such a solution, it is necessary to transform all pilocarpine to the protonated form by addition of HCI before the titration with NaOH. The titration curve will thua have two breake: the fmt for the excess of HCI and the second for pilocarpine hydrochloride. It should be pointed out that the degradation products of pilocarpinepilocarpic acid, i s o p i b a m i n e , and isoDilocarpic acid are codetermined in this titration; cf. ref IO. Titration Vessel. The total internal volume of the evedrop container in Figure 2 is about 800 FL, the internaldimensions of the container body being 5 X 5 mm. By opening the pipet in the bottom rather than by cutting off the tip, practically no container volume is lost. By use of this a p proach, there should be a minimum loas of sample solution sticking to the inside wall of the pipet BS a thin film. With a 3-mm-0.d. combination micro pH electrode the minimum sample volume is about 200 pL. Since the electrode and Teflon tubing occupy a volume of about 200 pL, the maximum volume of titrant is thus about 400 pL. Titration Parameters. The Teflon buret tip was firat placed 6-10 mm below the electrode tip, and the titrant was delivered near the narrow bottom of the vessel; cf., Figure 2. This approach gave poor mixing of sample and titrant, which resulted in noisy titration curves. The buret tip was therefore
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
A
PH
B
PH
12
12
IO
10
8
E
6
E
4
4
1
2 I
I
loo
I
I
Mo
I
I
300
pL 0.1 Y NaOH
pL 0.1 I NaOH
1589
4
0
,
,
loo
,
,
1
200
pL 0.1 M NaOH
Figure 3. Titration of pilocarpine hydrochloride in the eye-drop pipets at different stlrring and titration speeds, the latter being expressed as a criterion for electrode drift: (A) 67 rpm and 7.5 mV/min, (B) 200 rpm and 7.5 mV/min, and (C) 200 rpm and 3.75 mV/min.
Table I. Titration of Known Amounts of Pilocarpine Hydrochloride Using the Single-Dose Pipets as Titration Vessels amt o f pilocarpine HCl,
mg
sample
vo1,o PL
taken
found
difference, %
222 226 243 244 254 263
4.46 4.54 4.89 4.91 5.10 5.29
4.54 4.52 4.91 4.98 5.10 5.33
+l.8 -0.4 +0.4 +1.5 k0 +0.8
a Calculated from weight a n d density of the sample solution w i t h a known content of DilocarDine hvdrochloride.
moved higher up the electrode stem and a second Teflon tube was attached to the electrode as a “stirrer”; cf., the Experimental Section. Figure 3 illustrates the optimization of stirring and titration speeds. The stirring speeds are only approximate; they were calculated from a specified motor speed of 4000 rpm and the gearing-down factor. Noisy curves were obtained at low stirring speeds (A) and at high titration speeds (B), indicating incomplete mixing of titrant and sample solution. A stirring speed of about 200 rpm and a drift criterion of 3.75 mV/min gave smooth titration curves ( C ) ,and these parameters were therefore used in the rest of this study. A titration takes about 30 min, but the use of a more efficient stationary stirrer (e.g., a square or triangle-shaped electrode tip) might reduce this time considerably. Method Validation. The accuracy and precision of the titration method were determined by adding known amounts of pilocarpine hydrochloride to empty single-dose pipets and then carrying out the assay according to the procedure in the Experimental Section. The results are presented in Table I and correspond to an accuracy of +0.7% and a relative standard deviation of 0.8% ( n = 6). Pilocarpine Hydrochloride Assay. The pilocarpine eye drops 2% single-dose pipets are manufactured by the “bottlepack-aseptic system” (13)in sheets of 10 pipets. The determination of pilocarpine hydrochloride in the individual pipets of such a sheet gave a mean content of 4.83 mg of
pilocarpine hydrochloride/pipet with a relative standard deviation of 1.4% ( n = 10). Acidity Constant. The following values were obtained for the pKa of the pilocarpinium ion: 7.13 f 0.015 (multiple-point evaluation) and 7.13 f 0.018 (single-point evaluation). The results are the average of 10 determinations, and the uncertainties are the standard deviation. In the multiple-point evaluation, only data within 3-70% titration of pilocarpine HC1 were used. Above this interval, the pKa value increased from 7.2 to 7.4, indicating nonequilibrium conditions. This occurred when the volume of titrant exceeded 200 HLand is probably due to a slower mixing of sample and titrant when the vessel becomes full. Earlier single-point determinations of the acidity constant for the pilocarpinium ion, using normal-sized combination pH electcodes and 40-mL sample volumes, gave a pKa value of 7.15 (14). Since the acidity constant determined by microtitration agrees very well with the constant obtained by macrotitration, this indicates that equilibrium can be attained in very small titration vessels by use of the proposed stirrer design. Registry No. Pilocarpine hydrochloride, 54-71-7;pilocarpine, 292-13-7.
LITERATURE CITED (1) “United States Pharmacopeia”, 21st revision: Unlted States Pharmacopeia1 Convention, Inc.:, Rockvllle, MD. 1985; p 1277. (2) Albert, A.; Serjeant, E. P. The Determination of Ionization Constants”, 3rd ed.; Chapman and Hall: London, 1984; Chapters 2 and 3. (3) Schafer, H. Metrohm Inf. 1983, 73(1),5-6. (4) Walsby, J. R. Anal. Chem. 1973, 45, 2445-2446. (5) Spokane, R. 8.; Brill, R. V.; Gill, S. J. Anal. Biochem. 1980, 109, 449-453. (6) Steel, A. W.; Hieftje, G. M. Anal. Chem. 1984, 56, 2884-2886. (7) Gllgen, P.; Kobler, H. Chlmia 1978, 3 2 , 302-310. (8) Ebel, S.; Reyer, B. Fresenius’ 2.Anal. Chem. 1882, 312, 346-351. (9) Anonymous. Metrohm. Inf. 1980, 10(2),3-10. (10) Chung, P.-H.; Chin, T.-F.; Lach, J. L. J . fharm. Scl. 1970, 59, 1300-1305. (11) Johansson, P.-A.; Stefansson, U.: Hoffmann, G. Anal. Chim. Acta 1983, 151, 49-63. (12) Gibbs, I. S.; Tuckerman, M. M. J . fharm. Sci. 1974, 6 3 , 276-280. (13) Zlmmermann, L. fharm. Ind. 1983, 45, 1175-1181. (14) Johansson, P.-A.; Thelander, S. “Astra Pharmaceutical Production AB”, unpublished results, 1984.
RECEIVED for review November 18,1985. Accepted January 9, 1986.
