High Throughput HPLC Analysis of Barbiturates Application Note
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High Throughput HPLC Analysis of Barbiturates Application Note Forensic Toxicology Author Introduction John W. Henderson, Jr., Cliff Woodward, and Ronald E. Majors Although hundreds of barbiturates have been Agilent Technologies, Inc. synthesized over the past century, only about a 2850 Centerville Road dozen are used today. One factor deter-mining Wilmington, DE 19808-1610 which barbiturate is prescribed depends on the USA duration of its effectiveness. Duration of effect is determined by chemical struc- Abstract ture and this depends mainly on the alkyl groups attached to carbon #5 (see Figure 1) which confer In this study, an established HPLC method for USP Pheno- lipid solubility to the drug. The duration of effec- barbital was sequentially and quickly improved to a high tive action decreases as the total number of car- throughput method using Rapid Resolution (RR) and Rapid bons on C #5 increases. To be more specific, a long Resolution High Throughput (RRHT) techniques. New duration effect is achieved by a short chain and/or RRHT column technology is an especially powerful tool for phenyl group. A short duration effect occurs when improving lab productivity by reducing HPLC analysis time there are the many carbons and branches on the dramatically. Starting with a proven rugged method, alkyl chain [1]. conversion to a high throughput method can be achieved simply by replacing the original analytical-sized column Fast analysis of blood and/or other body fluids or with either a RR or RRHT column. The reasons for this hair can be useful in emergency situations. Figure direct conversion include the similar selectivity of smaller 1 lists the chemical structures of the barbiturates 1.8- and 3.5-µm particles compared to larger used in this study. 5-µm particles, and an engineered particle size distribu- tion that reduces unacceptably high system pressures that may be experienced with other sub-two micron packings. O O O CH CH CH HN NH 2 2 HN NH NH O O CH2CH CH2 O O CH3 O N O H R groups attached to the number 5 carbon atom Barbituric acid Phenobarbital Allobarbital CH3 O O CH2CHCH3 3CH N NH CH3 CH2CH CH2 OON OON H H Butalbital Hexobarbital Figure 1. Structures of barbiturates used in this study and barbituric acid. Start with a Proven HPLC Method They maintain column efficiency and resolution because the particle size decreases in proportion The objective of this investigation was to develop a to the column length. To demonstrate the scalabil- high throughput HPLC method for the analysis of ity of three different column lengths and particle barbiturates based on an existing slower conven- sizes for the test barbiturates, Figure 2 shows an tional HPLC method. Rapid Resolution (RR) and overlay of three chromatograms. The top chro- Rapid Resolution High Throughput (RRHT) HPLC matogram is the original USP method for pheno- column technology allows one to convert the exist- barbital with the internal standard (caffeine) but ing method into a high throughput method easily with the three additional barbiturates. The USP and straightforwardly to provide a gain in method specifies a 4.6-mm × 250-mm column with productivity. L1 type stationary phase (C18 phase bonded to silica particles) with a minimum resolution of 1.2 For established pharmaceuticals, the starting point [ ]. A ZORBAX Eclipse XDB-C18 column with these for HPLC methods is the United States Pharma- 2 dimensions and 5 µm particles was selected for this copoeia 27 (USP). The USP method for phenobarbi- separation and produced an analysis time of about tal can be found in reference 3. In addition to the 32 minutes. Simply replacing this column for a long acting barbiturate, phenobarbital, we added shorter one, 4.6-mm 100-mm with 3.5 µm an ultra-short (hexobarbital), a short (allobarbital), × particles, reduced analysis time by about a factor and an intermediate (butalbital) acting barbiturate of 2.5 (or 13 min), or by a factor of five using a as standards to demonstrate the power of RR and 4.6-mm 50-mm column with 1.8-µm particles (or RRHT columns. × 7 min). These latter columns provided faster sepa- rations with only a small loss of resolution and are Scalability of Barbiturate Reversed-Phase HPLC Method referred to as RR and RRHT columns, respectively. Scalability refers to the ability of an HPLC method These data show that older methods done on larger to use columns of different diameters and/or particle columns can be easily transferred to RR lengths and particle sizes and still maintain the and RRHT columns without sacrificing separation separation characteristics of the method. Earlier, it integrity. As can be noted in Figure 2, the resolu- was demonstrated that ZORBAX columns with tion of both the 3.5- and the 1.8-µm columns is smaller particles and shorter lengths provide simi- easily within the minimum specification of the USP lar chromatography in a fraction of the time com- method. pared with longer columns and larger particles [3]. 2 Eclipse XDB-C18 1 4.6 × 250 mm, 5 µm mAU P/N 990967-902 60 32 min. Pressure = 174 bar R2,3 = 5.99 40 R4,5 = 4.43 3 20 2 5 4 0 51015202530min Eclipse XDB-C18 4.6 × 100 mm, 3.5 µm (Rapid Resolution, RR) mAU P/N 961967-902 1 Caffeine (ISTD) 40 Pressure = 167 bar 2 Allobarbital 30 13 min. 3 Phenobarbital 20 R2,3 = 4.88 R4,5 = 3.72 4 Butalbital 10 5 Hexobarbital 0 Mobile phase: 60% A, 40% B 51015 A = pH 4.5 Na Acetate B = MeOH Eclipse XDB-C18 Flow: 1 mL/min 4.6 × 50 mm, 1.8 µm (Rapid Resolution HT, RRHT) Injection volume: 2 µL mAU P/N 927975-902 Detector: UV 254 nm 30 Pressure = 291 bar Flow cell: 2 µL, 3 mm flow path 20 R = 4.59 2,3 R = 3.32 10 4,5 7 min. 0 5 Figure 2. RR and RRHT column configurations increase speed and maintain sufficient resolution. Stationary Phase Selectivity as a Function of Particle Size Figure 3 depicts an overlay of the high throughput separation of barbiturates by equivalent 4.6-mm × The productivity increase by changing column 50-mm Eclipse XDB-C18 columns packed with dif- dimensions and particle size is achieved due to the ferent particle sizes. Note selectivity (α) is the reproducibility of the spherical silica particles and same for all three columns, independent of particle bonded phase. The highly uniform manufacturing size. The same selectivity indicates the three differ- process of the ZORBAX base silica, of the organosi- ent sized particles are chemically very similar. The lane bonding process, and of the standardized test- different sized particles can be packed in different ing of the column leads to the ability of the user to column dimensions with predictable and scalable freely substitute columns without suffering selec- results, because they exhibit the same chromato- tivity changes which would ultimately affect reso- graphic characteristics. Figure 3 also demonstrates lution. One way to measure this uniformity is to that as particle size decreases, efficiency increases. compare the selectivity factors among different Note that peak widths decrease, producing better particle sizes. Selectivity (α) is the ratio of the sensitivity (taller peaks), as the particle size retention factors: α2,1 = k'2/k'1 where k'1 is the decreases from 5 to 3.5 to 1.8 µm. adjusted capacity factor for compound 1 and k'2 is the adjusted capacity factor for compound 2. 3 1 t0 = 0.44 min 5 µm α3.2 = 1.20 α4.3 = 1.97 α5.4 = 1.13 P = 73 bar 5 2 3 4 123456min 1 t0 = 0.44 min α α α 3.5 µm 3.2 = 1.20 4.3 = 1.97 5.4 = 1.14 P = 111 bar 2 3 5 4 123456min 1 t0 = 0.46 min α = 1.22 α = 1.94 α = 1.16 1.8 µm 3.2 4.3 5.4 P = 291 bar 2 3 5 4 123456min Columns: Eclipse XDB-C18, 4.6 x 50 mm Peak Widths (min) Mobile phase: 60% A, 40% B 5 µm 3.5 µm 1.8 µm A = pH 4.5 Na Acetate 1 Caffeine (ISTD) 0.043 0.033 0.028 B = MeOH 2 Allobarbital 0.097 0.073 0.058 Flow rate: 1 mL/min 3 Phenobarbital 0.116 0.089 0.072 Injection volume: 2 µL 4 Butalbital 0.213 0.165 0.138 Detector: UV 254 nm 5 Hexobarbital 0.237 0.187 0.167 Flow cell: 2 µL, 3 mm flow path Figure 3. Particle size influence on selectivity and peak width. Optimization of Resolution with RRHT technology diffusion. At lower flow velocities, the contribution from axial diffusion to the H value may be quite A van Deemter plot is often used to depict the high and analysis time quite long. The “C” term changes in column efficiency, usually expressed as relates solute mass transfer from the mobile phase H (or Height Equivalent to a Theoretical Plate, to the stationary phase and vice-versa. It is both HETP), as a function of linear velocity (propor- flow rate and particle size dependent. For a typical tional to flow rate). A typical van Deemter plot is column packed with larger particles, say 5- or shown in Figure 4a. 10-µm, the column will behave like the van Deemter plot in Figure 4a. However, for smaller The shape of the plot can be described by Equation 1: particles such as the 1.8-µm packings, the slope of H = A + B/u + Cu (Equation 1) the van Deemter curve at high linear velocities is much flatter, as depicted in Figure 4b.