ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978 (7) J. P. Riley
"Chemical Oceanography,"Voi. 2, J. P. Riley and G. Skirrow. Ed., Academic Press, London and New York. 1965, pp 378-381, 384. (8) E. Konig, Coord. Chem. Rev., 3, 471-495 (1968).
RECEIVED for review September 6, 1977. Accepted January
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3,1978. This work was supported by New York State Contract No. C108738 and by grants from Nassau and Suffolk Counties in New York State. New York Ocean Science Laboratory Contribution No. 83.
Determination of Phentolamine in Blood and Urine by High Performance Liquid Chromatography Frederic de Bros" Anesthesia Laboratory of Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02 1 14
Ernest M. Wolshin' Astra Pharmaceutical Laboratory of Clinical Pharmacology, St. Vincent Hospital, Worcester, Massachusetts
A high performance liquid chromatographic method for the analysis of phentolamine in blood and urine is described. The procedure requires 1 mL of biological fluids and involves the addition of a double internal standard, alkalinization, extraction into ether, followed by back extraction into sulfuric acid. Separation of drug and internal standards is accomplished by HPLC on a reverse phase column with octane sulfonic acid in the mobile phase as an ion pairing reagent. Chromatographfc separation is complete in less than 12 minutes. The assay is linear for concentrations from 15 to 5000 ng/mL. The limit of detection is 15 ng/mL for a 1-mL sample. Relative standard deviations for replicate samples average 4.57 YO,The assay Is specific for phentolamine. There is no interference from commonly coadministered drugs.
is time consuming but may be of value for monitoring of drug concentrations in urine. Although gats-liquid chromatography is employed to assess the purity of pharmaceutical preparations and possibly may be adaptable t o clinical determinations, the technique for derivatization of phentolamine and its measurement in nanogram quantities has not been reported. A rapid and specific, clinically applicable assay with nanogram sensitivity for phentolamine has not previously been published. This paper presents a n analytic method which meets the above criteria for phentolamine in biological fluids. I t can measure the drug in a broad concentration range and is suitable for pharmaceutical development purposes as well as drug monitoring in a clinical laboratory.
EXPERIMENTAL Reagents and Solvents. Phentolamine mesylate was provided by Ciba-Geigy, Summit, N.J. Antazoline base and naphazoline
Phentolamine o r 2-(N'-p-tolyl-N'-n-hydroxyphenylaminomethy1)imidazoline (Figure 1) is a n LY adrenergic blocking agent (1-5) with a slight @ stimulating effect (6). I t was introduced in 1950 ( 7 ) as a vasodilating drug for intravenous and oral administration (8). I t has been reported as a useful agent for therapy in congestive heart failure, myocardial infarction, arrhythmia, angina pectoris, shock, and bronchial asthma (9). Current pharmacologic investigations of phentolamine have been summarized in t h e proceedings of a symposium (10). Because of t h e recent interest for use of this drug as a continuous infusion or for long-term therapy, a simple, specific, quantitative assay in biological media is desirable. Techniques applied to t h e analysis of pharmaceutical preparations include titrimetry ( 2 ) , gravimetry ( I ) , UV spectrophotometry ( 1I), colorimetry (12-14), gas chromatography (15),and high pressure liquid chromatography (16). T h e colorimetric reaction (17) has been adapted for use in biological samples (18),but was not found to be specific for phentolamine. T h e HPLC analysis of phentolamine utilizes an ion-exchange column (16);however, the method is characterized by asymmetric peaks and poor resolution ( R < 1.0). Analysis of imidazoline compounds in biological samples with T L C (19) is specific for phentolamine, but is limited to quantities greater than 1 pg. In addition, the TLC technique 'Present address, Astra Pharmaceutical Products, Inc., Framingham, Mass. 01701. 0003-2700/78/0350-0521$01.OO/O
hydrochloride were obtained from Pfaltz and Bauer, Stanford, Conn. Sodium octane sulfonic acid was purchased from Eastman Chemicals, Rochester, N.Y. Triple distilled methanol, diethyl ether, and cyclohexane were obtained from Burdick and Jackson Laboratories, Muskegon, Mich. Distilled, deionized, neutrai, charcoal filtered, and bacteria free water was used for all solutions. All other reagents were analytical grade or better. Apparatus. A liquid chromatograph with a Milton Roy Minipump (Milton Roy Company, Riviera Beach, Fla.) was used. A pulseless solvent flow was obtained with a T configuration of dampers and restrictors (Waters Associates, Milford, Mass.). Pressure was monitored continuously with a glycerine filled, 5000 psig Lenz gauge through a high pressure manifold connected to a 10000 psig Circle Seal adjustable relief valve, set for a 5000-psig cracking pressure. The mobile phase was continuously filtered through the solvent inlet line by a 30-pm filter (No. 25531, Waters Associates). Further particulate matter was removed from the solvent by a 2- and 0.5-pm filter (Swagelok SS2F-2 and SS2000-SR12, respectively) placed in series after the pulse damping network. Samples were injected through a 7OOO-psig, six-port Valco valve, with a 50-pL loop. A Glenco sample injection syringe (Model VIS 50-700) and filling port (VISF-1) were used. Separations were accomplished on a Microbondapak/CI8 column (U'aters Associates) maintained at constant temperature in a 20-L water bath. The drug concentration in the colunin effluent was monitored by a variable wavelength detector (model SF770, Schoeffel Instruments. Westwood, N.J.). Chromatograms were recorded with a model 252A Linear Instruments strip chart recorder. Chromatographic Conditions. Separations were performed at 2.4 mL/min and at a back pressure of 4000 psig. The column 1978 American Chemical
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978 R
Table I. Recoveries of Naphazoline (N), Phentolamine (P), and Antazoline ( A ) from Blood and Urine N P P P A
N A N - "
W R
COMPOUND ANTAZOLINE
-cH2B
NAPHAZOLINE
PHENTOLAMINE
OH
Figure 1.
Imidazolines and their generic names
was maintained at 25 i 0.05O C and the effluent was monitored at 280 nm. Stock Solutions. Phentolamine stock solution was prepared by dissolving 5 mg of the mesylate salt in 100 mL of distilled deionized water. The solution was used for preparation of standard curves and absolute recovery studies. The internal standard solution contained 50 Fg/mL antazoline base and 1 pg/mL naphazoline HC1 in water. All stock solutions were stored a t 4 "C in the dark. Mobile Phase. The mobile phase was prepared from a methanol and an aqueous component. Each component contained 6.16 mM 1-octane sulfonic acid and 170v/v glacial acetic acid. The methanol solution was adjusted t o pH 4.0 with 1.0 N potassium hydroxide in methanol and the aqueous solution was adjusted to the same pH with 5.0 N sodium hydroxide in water. One percent acetonitrile was added to the aqueous component as a preservative. The mobile phase solution was made by adding 52 parts of the methanolic component to 48 parts of the aqueous component. Extraction Procedure. The extraction was performed by pipetting 1.0 mL of blood or urine, 100 pL of internal standard solution, 500 p L of 1.0 M ammonium hydroxide, and 6.0 mL of water-saturated ether into a W m L , conical bottom, centrifuge tube. After capping the tube with a polyethylene stODper, it was mixed for 20 min at 18 rpm on a Drummond Scient.fic Rotary Mixer. The organic and aqueous phases were separated by centrifugation of 20 min at 1500 rpm (400 X g). Five milliliters of the water-saturated ether were transferred to a clean 15-mL centrifuge tube and mixed with 25 pL of 0.2 M sulfuric acid, by vortexing for 1 min. The ether and sulfuric acid were separated by the addition and mixing of 6 mL of cyclohexane, followed by centrifugation for 20 min at 1500 rpm (400 x g ) . As much as possible of the cyclohexane-ether layer was aspirated off and the sample was warmed for 5 min at 70 "C in a water bath to eliminate residual organic solvent. The recovered aqueous volume (45-50 ILL)was deposited in the sample loop for injection. S t a n d a r d Curves. Samples for the standard curves in the 250 to 5000 ng/mL range were prepared from drug-free blood, or urine, by the addition of microliter volumes of the phentolamine mesylate stock solution (50 pg/mL). For samples in the 15 to 250 ng/mL range, the stock solution was diluted to 5 @g/mL. Study of Blood Concentrations. A 60-kg patient undergoing surgery for a pheochromocytoma received a 30-mg intravenous "bolus" injection of phentolamine mesylate (Regitine, Ciba-Geigy). Samples of arterial blood were drawn at the appropriate times and stored a t 4 "C in heparinized vacutainers for 18 h prior t o analysis.
RESULTS A N D D I S C U S S I O N When blood and urine samples were alkalinized with ammonia hydroxide, phentolamine could be extracted by using water saturated ether. The average p H of extraction was 10.3.
medium added pgrecovered,E
blood
blood
0.1 0.081
0.05
blood 0.5
urine
blood
5 3.2 0.041 0.389 4.23 SD,n=4 0.0092 0.0065 0.0833 0.645 0.452 RDS,% 11 21 15 14 16 Mean recovery, % 81 82 78 85 64 pig
5
Ether yielded a macroscopically clean phase after extraction and had a reasonably equal affinity for the drugs of interest. Direct evaporation of the ether extract gave residues which were insoluble in small volumes (
