CLINICAL CHEMISTRY
High-Performance Liquid Chromatography (Direct Injection Techniques) David J. Anderson Department of Chemistry, Cleveland State Uniuersity, Cleveland, Ohio 44115 The previous renew of high-performance liquid chromatography (HPLC) in clinical chemistry appearing in the applications renew of Analytical Chemistry (NI) covered the area of detection methods in HPLC, reviewing the topics of electrochemical and mass spectrometric detection. The area covered in this review will be HPLC separation methodologies, specifically reviewing the topic of direct injection techniques. Direct injection techniques are reviewed comprehensively, covering the period up to a Chemical Abstract date of October 1, 1992.
SAMPLE PREPARATION Overview. Sam lepreparation isavitalfmtstepin HPLC clinical analysis. he degree of sample preparation ranges from a minimal amount, as is the case for direct injection techniques, 10 being extremely labor intensive, as is the case for liquid extraction and precipitation techniques. Sample preparation (a topic in which direct in'ection techniques is included) serves one or more of the foliowing purposes: (1) it separates analyte from protein, such that the total compoundcan bedetermined(thefreeand theprotein bound), and such that theexposure of theanalytical column toprotein is minimal: 12) it removes interferences that either affect the accuracy of quantitation, affect retention of analyte on the column.and orextend analysistime;and. ort3)it concentrates the analytew of interest to improvesensitivity and detection limit capabilities. Reviews of sample preparation for H1'I.C in clinical analysis fur general rN2). biopolymer (N3-NSJ. and drug (.W, N n analysis have been written. Although precolumn derivitization is a very important technique in sample preparation, for the purposes of this review it is considered a detection enhancing technique and thus will not be covered. Detrimental Effects of Protein on Columns. Samples include bodyfluids(suchasserum,plasma,wholeblood,urine, cerebrospinal fluid, etc.1 tissue samples, and other samples, such as hair. This review will focus on sample pre aration methodologies used in HPLC analysis of serum antplasma samples. and unless specified otherwise, samples mentioned below are either serum or plasma. Direct injection of plasma or serum samples isespeciallydetrimentalto chromatographic columns (reversed phase and normal phase, which utilize mobile phases containing 1 5 5 or greater organic modifiers (NR,. because these samplescontain large amounts of proteins, which are precipitated and or denatured and subsequently adsorbed onto the packing material. leading to bark-pressure huildup. chan es in retention time, decreased column efficiency, and cfe'efreased column capacity (N9-Nll1. The decreased efficiency probably results from the denatured protein inhihiting diffusional mass transport of the analyte to the packing material surface (Nllr. It should be noted that significant precipitation of proteins leading to great increase in back pressure after only a few injections requires at least 70Crmethanol content in the mobile phase (NIGJ.A study in which precipitation of serum proteins was measured by a light-scattering experiment found precipitation cutoffs for percent urganic mudifiers of 2S0i fur acetonitrile, 20'; for 2-propanol, and I O 7 for tetrahydrofuran. with the remainder of themobile phase beingO.l M phosphate buffer, pH6.8 r.VI&. Anotherconcernisthemobile-phase pH, with precipitation occurrin at the protein's pl. The range of pls and concentrationsof t t e various electrophoresis serum bands are as follows: albumin (4.7, 45 mg mlA: ol-globulins (1.85.2,6 mg mLJ: 02-globulins( 3 . S 5 . 2 , 9 mg mlA; d-globulins (5.3-5.9, 11 m ,mid: and -r-globulins (6.3-7.3, 15 mg mLJ (.V13. N14). ldditionally, for plasma samples, fibrinogen has a pl of 5.5 ( . Y l 5 ~with a normal concentration of 2-4 mg mL (Nlfi). Althoughallreversed-phasepacking material is susceptible to the adverse affects of proteins, wide-pore supportn. which have large enough pores to allow the passage of protein molecules into them, show particularly augmentrd adverse effects (.Via.
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D a w J. A n d m was me overall COOTdinator for mis review and me coordinator 01 me wrmng of the lnsbumental sectlon. he received his B S In cnernlstrv lrom Buckneil Universlty In 1977 and hi; P h i . In analylical chemistry from Iowa Slate Universltyin 1966. From 198610 1968h e was a postdoctoral fellow In clinical chem Istry at the Mayo Cllnlc. He Is currently an ASSIstant Professorand Director of Clinical Chemistry at Cleveland State University. He isalsoa Dlpbmatofme American Board of Clinical Chemistry. His research Interests are the use of HPLC methodology In me dagnosls of d sease He 1s curenliy developing nigh-performance anlnq chronwl~raphictechniques for measurmg new m a r w r s lor tnromoosls and cancer. as well as developing hPLC techniques #n lne delermlnatlon 01 soenzymes In card ouascular disease and inhernea a sorders ~
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Classical methods of sample preparation include precipitationof proteins and/or extractionofanalytes intoorganic phases (liquid-liquid extraction). Liquid-liquid extraction is the most common method for sample preparation which serves the dual function of sample cleanup (to eliminate interfering compounds) and of deproteinization (to remove analyte from the protein matrix). These methods are labor intensive and time consuming. They also require a large volumeofsample(at1east 1mL),aswellasadditionofinternal standard (due to analyte loss resulting from the multiple sample manipulations). In precipitation methods, precipitating agents (organic modifiers and acid agenta such as trichloroacetic, perchloric, tungstic, and metaphosphoric acids) are added to the sample and the precipitate is removed by centrifugation. The disadvantages of precipitation methods are increased total analysis time and reduced recovery due to adsorption of analyte onto the precipitated protein. As an example, in one work using trichloroacetic acid precipitation, 67-78% recoveries were found for the low control of theophylline and its metabolites (N18).
INTRODUCTION TO DIRECT INJECTION TECHNIQUES Direct injection of samples onto HPLC columns is substantially advantageous in the clinical laboratories in terms of ita time- and labor-savin capabilities, in addition to other advantages given below. geveral direct injection methods have been devised which deal with the problem of protein being present in the sample. The methods include the precolumn technique, restricted access media, and chromatography in mobile phases containing surfactant. Highperformance affinitychromatography is alsoa direct injection technique; however, it will not be covered in this review. The characteristicaandperformanceofeachdirectinjection technique are comprehensively discussed below for the analysis of serum and plasma samples. Astoundingly, direct injection techniques have even been devised for the direct injection of blood samples onto the HPLC in the determination of the drugs carbamazepine, procainamide, N-acetylprocainamide. and chlorpromazine (N19-NZl) and in the analvsis of adenosine and adenine nucleotides (N2.2). In this ~~~~~~~~~~~~,~ met6od a precolumn terhnique is used, ha\,ingthe capability of directly injecting I 0 0 whole blood samples (1&200-rL volume) without back-pressure buildup (althoughforthe200pL injections, rinsing of end fittings with 0.1 M NaOH and 50% methanol under sonication was done whenever hackpressure prohlemswere noted) (NlSN21). Reviews ofdirect injection techniques have been published (N.23, N24). The only preparation step for direct injection techniques of serum or plasma is centrifugation at 3oo(t5000g for about 10 min (NIO,N25, NZfi), although 10 OOO rpm was found to benecessarytopreventcloggingofthefritinonestudy(N27) and l2OOOg for 5 min was required in another study (N28). ~
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In another study, visual inspection of thawed serum samples was done, and centrifugation at 2000 waa performed only if particulate matter was noted (N29). tentrifugation is better than filtration because there is minimal loss of sample, and it is cost saving (since disposable supplies are not used).
PRECOLUMN TECHNIQUES Description. The precolumn technique is the direct in'ection technique that is most reported in the literature. The precolumn technique utilizes two columns in series ( recolumn and analytical) connected by a switching valve. l ! he most common precolumn technique employs a reversedphase precolumn and a reversed-phase analytical column. In the most common case, sample is injected into a ueous mobile phase flowing through a precolumn (1-4 cm inlength, 4-4.6mm i.d.) which retains lipophilic compounds, paasing nonretained hydrophilic compounds such as proteins to waste. The switchin valve is then changed and components retained on the precofumn are eluted onto the analytical column by increasing the strength of the mobile phase. This technique serves the dual function of concentration of analyte and removal of rotein. A review has been published covering the practicJand technical aspects of this methodology (N30). Advantages and Disadvantages. There are many ad'an?. es of the precolumn injection technique in comparison to tra itional sample preparation techniques. The precolumn techni ue saves time in comparison to the labor-intensive liquidliquid extraction and recipitation techniques. Also less sample is required [usua rly 1mL or more is required for liquid-liquid extraction techniques (N31)I and better recoveries are obtained [110% for precolumn, 73% for liquidliquid extraction for a metabolite of urapidil (N31)l. One study, however, showed liquid-liquid extraction procedures to be more precise (2-3 times) at lower concentrations of analyte (urapidil and ita metabolites) (N31). Precolumn techniques have significant advantages over solid-phase extraction (SPE) techniques. Besides being less time consuming, the recolumn technique is less costly with respect to supplies. everal hundred sample in'ections can be made on the precolumn, in comparison to the APE column (which is disposed of after one sample). There are other advantages over the SPE technique. The technique is highly reproducibleand has high recoveries and thus does not require the addition of an internal standard (N32). An internal standard technique can be less precise than external calibration methods (N33), as well as having the difficulties associated with finding an ap ropriate compound to serve as an internal standard. In the &termination of desferoxamine and ferrioxamine, the precolumn technique had better recovery, reproducibility, and faster analysis time than the SPE method (N34). In the determination of tricyclic antidepressants there was a potentially interfering exogenous peak in the SPE technique that was not present in the precolumn technique (N35). An advantage of the precolumn technique over other direct injection techniques is ita superior detection limit capabilities due to ita allowance for injection of large sample volumes. In one study 1.0 mL of diluted plasma (1mL of plasma and 0.2 mL of acetonitrile) was in'ected for the determination of retinoids, with a detection /i.mit .of 0.3-0.5 ng/mL using UV detection (N36). The detection limit capabihty was found to be 0.2 ng/mL for amiloride in plasma using fluorometric detection, which is a 5-fold improvement over the SPE technique (N37). Precolumns have also been used in microbore chromatography to increase sensitivity, despite some decrease in system efficiency. This was demonstrated in the determination of diazepam in serum in which a 15 mm X 3.2 mm (6-pm) Cle precolumn was used in conjunction with a 250 mm x 1 mm analytical Cle column (5 pm), allowing for injection of 1 mL of serum, which produced a 1500-fold increase in sensitivit while causing only a 15% decrease in system efficiency Analytss that are unstable and require speed anal ais are best done by direct injection techniques, such' as t i e precolumn technique, as exemplified by the analysis of doxorubicin and daunorubicin (N27) and of photosensitive retinoids (N36). In some cases, sample preparation causes inaccuracies due to chemical conversion, which does not happen in direct injection techniques (N39). The advantage
8
(db.
of the precolumn technique is also exemplified in the determination of folate monoglutamate, which is sensitive to oxidation in heat and chemical precipitation methods (N40). The precolumn technique also cuts analysis time by not allowing strongly retained components onto the analytical column. This is accomplished by "cutting" analyte peaks from the precolumn onto the analytical column through appropriate timing of the switchin valve (N30, N41). This was demonstrated in the analysis o urinary vanillylmandelic acid and homovanillic acid (N42),and in the analysis of a calcium antagonist drug, in which the run time was shortened to 25 min, with the elimination of late-eluting peaks at 60120 min (N43). The major disadvantage of the precolumn technique (unless the loop column technique is used, which is discussed below) is the need for an additional pump, a column switchingdevice, and timed computer control of events. Design and Operating Conditions. Precolumn Packing Materials. Most precolumn applications in clinical analysis have used reversed-phase precolumns with reversed- hase analytical columns. However, there have been ion exclange (N44) and size exclusion (N45, N46) precolumns used in conjunction with reversed-phase analytical columns and normal-phase precolumns used in con'unction with normalphase analytical columns (N47). d o i c e of the type of reversed-phase packing material to use in the precolumn is discussed later. System Design. Severalt es of column-switchingdesigns have been used. The back3ush design is most often used (N48) because it minimizes band broadening ( N l 0 , N 4 S N51). However someprefer the forward-flushmode to protect the analytical column from possible impurities at the head of the column (N34,N52). Another advantage of the forwardflush mode is the additional removal of interferences, as seen in one study measuring @-lactamantibiotics in urine, in which operation in the back-flush mode had interferences that were eliminated by timed column switching in the forward-flush mode (N50). Another study also recommended the use of the forward-flush mode, because it removed a significant number of interferences that were resent in the back-flush mode for injected serum samples (&3). Designs for forwardflush and back-flush of a precolumn using a six-way valve and two pumps are presented (N50). The typical precolumn direct injection design involves one precolumn, one analytical column, column switchingvalve(s), and two pumps, one to introduce the sample onto the precolumn directed to waste and the other to elute the bound species from the precolumn to the anal ical column for subsequent separation (N35). Some of the esignsalso include a guard column placed before the analytical column (N31). Use of an on-line 0.5-pm filter is recommended, which needs periodic replacement (N51,N52). A desi with two six-way valves has been used (N54-N56). Anotcr one-precolumn design allowscleaning of the precolumn with organic modifier solutions subsequent to flushing analyte to the analytical column (as the analyts is being chromatographed on the analytical column) (N54). This cleanu step for the precolumn extends the lifetime of the preco umn. A desi that incorporates a second precolumn parallel to the f i t !%been employed which increases sample throughput, by alternatin injection on one precolumn and backflushing of retainecfcompoundson the other precolumn (N57). Another design has two precolumns in series, which gives extra versatility in cleanup, allowing for injection of larger sample volumes because contamination of the analytical column is minimized (N58). This design was used for the determination of cortisol in serum and urine (N59). The loop column method is a one-pump precolumn method which does not require accessory equipment. This method is a manual injection method in which the injection loop of a manual injector is replaced by a precolumn, with the sample being injected and washed manually in the load position, rior to injection (N37, N51, N60). Loop injection has also een completely automated by use of an autosampler (NZS). Several desi s have been used in gradient work (N31, N36, N61). A Toxcar chromatograph approach using one precolumn produced an impressive tLoughput rate of 40 samples/h in the determination of primidone, phenobarbital, phenytoin, and carbamazepine in serum (N62).
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Table N-I.Effect of Particle Size of Packing Material in Precolumn on Chromatographic Performance after 100 Injections of 100 p L of Serum Analyzed for Primidone
(NW precolumn particle size (pm)
5 10 37-50 30
pressure incr (bars) preanal.
theor plates anal. column
reversed-phase berec material column column fore after (%) 13.6 27.2 7080 2600 100 SupelcosilCs 3.4 13.6 6305 3560 100 LiChrosorbCls 5 Corasil CIS 0 1.36 7400 7450 8325 7942 >90 6.8 0 LiChrosorbCls
Experimental Conditions: (a) Mobile phase (1) wash, aqueous separation, 4655 methanoV2.5 mM sodiumdihydrogenphosphate. (b)Columns (1)precolumn, 25 X 3.9 mm; (2) analytical, 150 X 4.6 mm, 5-pm eilica bonded with a
0.1 M phosphate (pH 3.5); (2)
dimethyloctylchlorosilane.
There are several design considerations for precolumns to minimize protein's adverse effects. Large-diameter packing material (25-50 pm) is most often used in precolumns. It has been shown that approximate1 2-3 times more injections can be made prior to pressure b d d u p problems on precolumns packed with 50-pm material compared to 10-pm material (N63). A systematic study of precolumn particle size on performance was done showing the effect of precolumn particle size on pressure buildup and column efficiency, clearly demonstratin -the advantage of larger size packing materid (see Table N-% (N62). The materid b e d ' i n end fittings of the precolumn is a critical aspect to lifetime in precolumn injection techniques. Steel frits appear to be the limiting factor in direct injection techniques, as it was reported that there is a loss of column efficienc and buildu of back pressure that was corrected after repracement of t i e frits [loss of efficiencywas seen after 100or more 10-pLserum in'ections were made on an internalsurface reversed-phase (ISkP) column (discussed later) with 20% acetonitrile, 0.1 M phosphate (pH 6.8) mobile phase, which was corrected by replacing the frits] (N13). It should be noted that the presence of organic modifier in the mobile phase greatly increases protein adsorption, as over loo0 (10 pL) injections of serum were done on the same ISRP column with minimal pressure buildu us' an aqueous mobile phase containing no organic modiler 313). Screens have been used in place of frits (N62,N64). There was a 38 % decrease in overall system plate number and an increase of 17.7 bars pressure across the analyticalcolumn for 75 (1OOpL)injections of serum for recolumns containing frits compared to no change in eitier back pressure or plate number with 100 injections of serum on columns containing screens (N62). Other end-fitting modifications have been employed, such as the use of Teflon frits with pore diameters of 15 pm (N48). Another work used sieves with 30-pm pore diametersto enclose the precolumn packing material for injection of large volumes of solubilized tissue samples (>30 mL) (N51). Optimization. Optimization in precolumn techni ues involves maximizin the percentage of analyte retainel on the precolumn whfe minimizing the retention of proteins and interfering compounds. Several chromatographic parameters need to be optimized with respect to the precolumn technique including the followin : precolumn packing material, precolumn length, mobile-p%awcomposition,injection volume, and flow rate as discussed below. Also relevant to optimization is the effect of anal binding to protein and the polarity of the analyte, whic is also discussed below. Precolumn Packing Material. As mentioned previously, most applications use a reversed- hase recolumn with a reversed-phase analytical column. Bever a rstudies have been done comparin the performance of various precolumn packing materi3s (N51, N52, N65, N66). It is important to note that different recoveries and/or retention capabilities were shown for precolumns acked with CISmaterial from differentmanufactures (N47,%52,N67),and thusthe packing material's source, in addition to the stationary phase's chemical composition, is critical to the proper choice of precolumn packing material. Strength of retention depends on the polarity of the analyte and packing material, with the
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
scale of packing material polarity being (highest to lowest polarity) diamino > amino > cyano > dimethylamino > nitro > diol > Cz> C8 > Cl8 (N48).In general, there is increased stren h of retention expected for hydro hobic drugs on preco umns containing packing material laving increased alkyl chain length. However, strength of retention is compound specific, as exemplified in the determination of rifampicin, which was found to be more strongly bound to shorter alkyl chain and phenyl packing materials compared to c18 packing material (N68).Use of less retentive precolWE, such 88 C2 (N31, N66)and cyano (N69, N70) with Cia analytical columns is advantageous from an interference standpoint, as long as the analyte is sufficiently retained by the precolumn. Use of a CZprecolumn was also found to be advantageous for decreasing analysis time, in that less time is needed to elute analyte from the precolumn (N31). For olar analytes, cyano and phenyl precolumns showed much Ess retention roperties and more band broadening than Ca and C18 preco umns (iV51). Protein-coated c18 precolumns have also been used (N26,N71-N74); however, there has been no study done that demonstrates an advantage of this precolumn over other precolumns. In fact, these columns would be expected to show poor efficiencies, since the protein treatment appears to partially block the pores (N71). Application Mobile Phase. In the context of this review, the ap lication mobile phase is the mobile phase pumped the precolumn when sample is injected and the ' k 3 u m n is washed. In special instances the wash step will Ee referred to separately. In most w e e sample is in'ected into an application mobile phase consistingof a ueoushffer or water pumped through the recolumn, withxydrophobic compounds being retained an$ protein passing through the precolumn to waste (N30,N49). An apphcation mobile phase consistingof an aqueous buffer at an ap ropriate pH to make the compound neutral is necessary for acidic and basic compounds. For example, a H 3phosphoric acid application mobile phase (along mth agdition of phosphoric acid to the plasma sample) was used for the retention of the acidic metabolite of the drug mefloquine on a Cle precolumn (N75). In another case, lowering the pH of the sample, in addition to the use of low H a phcation mobile phase, was necessary for retention or acisic compounds on a reversed-phase Note that phosphoric acid is used for pH precolumn "6). adjustment as opposed to perchloric or sulfuric acid, which causes protein precipitation. In other cases a low concentration of organic modifier is placed in the application mobile phase to remove interferences or to disrupt protein binding (N77). For determinations at low UV (230 nm) it was necessary to add organic modifier to the application mobile phase in order to eliminateasignifhnt interferingpeak which was present when just water was used in the mobile phase (N77). However, this may have also reduced recovery (7196%) (N77). At detector wavelen hs above 257 nm, however, there were no interfering pe noted with the water application mobile hase, and thus the addition of organic modifier to the spp&cation mobile phase was not necessary (N77, N78). In another study, an additional wash at lower pH eliminates many interferences, by eluting off matrix compounds, while maintaining the retention of the drug midodrine on the precolumn (N55).An apparatus has been used that allows for successive flushin? of precolumna with different eluants (N58).For application mobile phase not containing organic modifiers, the time of wash is not critical to optimization,as long as there is enough time for the proteins to pass through the precolumn. Longer wash times do not usually adversely affect chromatography,although sli ht band broadening may occur with extended wash time (835). Other Chromatographic Conditions. A theoretical work has been ublished estimatingthe maximum volume of sample that cane! injected onto a precolumn and still be within the limits of a 20% loss in resolution for the system (assumes back-flushing), as well as estimating the required column length of the precolumn (N79). The stronger the retention of the analyte on the precolumn the greater the amount of sample that can be injected onto the precolumn without loss of recovery, and thus the in'ection amount is determined for the least retained anal@ Experimental determination of the break-thro hvolume for the analytefor the application mobile phase u s 3 is essential to characterizing precolumn techniques (N80). The effect of flow rate through the
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CLINICAL CHEMISTRY
precolumn on recovery is particle size de endent, with no recovery loss for 37-44 pm, 1-2 5% recovery 08s for 63-74 pm, and 10% recovery loss for 125-149 pm sized packing material at 2 mL/min com ared to 0.5 mL/min flow rates for the drug methaqualone, wkch is 80% protein bound (N81).A study was done for prostaglandins comparing various precolumn stationary phases and lengths, studyin the effect of sample acidification, and comparing the precofumn technique m t h SPE (N82). Effect of Protein Binding on Recouery. Dr s having low protein-binding characteristics, such as primqone (0-10 % protein bound) to moderately hi h protein-bindingcapability, such as carbamaze ine (65-85 $, protein bound), have been effectively retainet! by reversed-phase precolumns with an aqueous (no organic modifier) application mobile phase (9195% recove of the drug) (N62).There have been problems however wix very tightly protein-bound compounds, as is the case for some retinoid metabolites, which showed poor recoveries no matter what application mobile-phase conditions were tried (N83). Adding acetonitrile to serum and plasma samples (total concentration of 10% acetonitrile in sam le) was found in one study to be sufficient to improve ow recoveries of drugs tightly bound to protein (50-70% recovery without acetonitrile, >95% with acetonitrile) (N51).In another study the surfactant sodium dodecyl sulfate (SDS) was added to the plasma sample to increase the recovery of teniposide from 8% to 90% (N84).It was noted that precipitation of protein occurs between SDS concentrations of 8.3 (which is the critical micelle concentration for SDS) and 35 mM, and thus addition of SDS to a final concentration of over 35 mM is recommended (N25, N84). Interestingly this criterion does not apply to a plication mobile phases, as it has been found that intermegate concentrations of SDS in application mobile phases (8-35 mM) are effective in solubilizing proteins in samplesthat are directlyinjected (N84, N85). Quantitative recoveryofthe retinqids 'equired-addition of acetonitrile to both sample and application mobile phase (N86); however, the retinoid etretinate required protein precipitation with ethanol, and even then recoveries were only 6 H 6 % (N87).Poor recoveries on reversed-phase precolumns are found articularly for acidic com ounds, which bind stronglyto altumin. This was addressed gy using a pH 3 application mobile phase in the determination of tryptophan metabolites, in which it was found that acidic tryptophan metabolites showed 100% recovery, compared to 4-17% recovery using a pH 7 ap lication mobile phase on a In this study a high protein-coated Cu recolumn recovery for nonaci8c tryptophan metabolites was also found when an acidic application mobile hase was used. A 0.05 M phosphoric acid application mob& phase (along with acidification of the sam le) was used for the determination of acidic antibiotics cermandole and cefamandole nafate by a precolumn direct injection techni ue of plasma and urine A combination aci! treatment and addition samples (N89). of acetonitrile (adjusting plasma sample to pH 3 and 5% acetonitrile), along with a pH 2.9 application mobile phase, was necessary for quantitative recovery (compared to 3550% recovery without sam le treatment and a pH 7.4 application mobile phase) oft e drugs N-l-leucyldoxorubicin and doxorubicin, which are both 75 % protein bound (N28). In another case, a very high methanol concentration (above 40% in the precolumn application mobile phase) was necessary for the recovery of nonpolar eicosanoids, which presented a problem in that olar eicosanoids show reduced recoveries at these methano concentrations, leading to an intermediate methanol concentration (15% ) being chosen for the initial application mobile phase and the need to use gradient elution in acetonitrile and methanol to separate the eicosanoids of differing polarity (N90). Hydrophilic Compounds. Direct injection precolumn techniques are more suited for the determination of hydrophobic compounds than hydrophilic compounds. Determination of h dro hilic compounds has been addressed by several woris. hedium- olarity precolumns (such as CZ, cyano, or diol) or normafphase columns (both precolumn and analytical) have been used (N47).Addition of an ionpairing agent to the application buffer has been done for the determination of tryptophan metabolites (N26) and p-ladam antibiotics (N50).In another approach, a hydrophobic interaction precolumn was used for on-line deproteinization
P
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of the sample. The application mobile phase was 0.4% erchloric acid, which caused adsorption of proteins and drophobic components onto the butyl precolumn, while owing ion-paired gentamicin (the ion-pairin ent was in the application and elution mobile phases, wf~% was necessary for retention of gentamicin on the analytical column) to ass through the precolumn onto a TSK gel 120-Aanalytical Absorbed proteins were then eluted from the cofumn (N91). precolumn (which was 50 mm X 4 mm, with a capacity for 250 pL of serum) to waste with a 12-min (flow rate 0.8 mL/ min) wash with 0.1 M phosphate buffer (pH 7) prior to the Hydrophobic interaction precolumns next injection (N91). have also been used in the determination of the hydrophilic drugs procainamide and N-acetyl rocainamide (N20) and adenosine and adenine nucleotides &22), with direct injection of blood or plasma in these analyses. For the determination of the basic compounds pholcodine and morphine in urine, a cation exchange precolumn was used (N44). Column Lifetime. Table N-I1provides a summary of the literature reporting column lifetimes for precolumn techniques. As recorded in Table N-11, some authors used exact criteria to assess the maximum amount of sample that could be injected before column replacement (either anal ical or precolumn), while others did not. The range of to&le (either plasma or serum) that was injected on the precolumn systems given in Table N-I1 before the analytical column required re lacing was 16-150 mL. In most cases there was a scheduletreplacement of precolumns, on-line filters, and/ or guard columns to attain the maximum lifetime for the analytical column. The precolumn system having a capacity of 150 mL employed 30-pm pore sieves in place of frits, underscoring their importance in direct injection techniques (N51).A particularly rugged technique used a size exclusion precolumn (N45).In this technique plasma samples were injected over a period of 1year without the need for replacing the precolumn. The disadvantages of using size exclusion precolumns, however, are increased interference (since all small molecules are introduced onto the analytical column) and poorer detection limit capabilities (sincethe size exclusion column does not concentrate the analyte). The lifetime of precolumns themselves as given in Table N-I1 was generally 20-30 mL of serum or plasma (for those studies in Table N-I1 which measured recolumn lietime). One study estimated the precolumn liztime to be approximately 60 mL of serum (N28). This high precolumn lifetime may have resulted from 3X dilution of sample (note a precolumn lifetime of 60 mL is given for undiluted sample), acidification, addition of acetonitrile to the sample, and a cleanup of the precolumn with 70% acetonitrile prior to the next injection. The lifetime of the precolumn system was found to decrease with increased or anic modifier present in the application mobile phase [40$0/8,20%/30,10%/80,5~/130;methanol percentage/number of consecutive injections of 10 p L of plasma before a eater than 10% increase of plate height was observed (80)l. Flow rates were also found to be important for system stability when plasma samples are injected into application mobile phases containing organic modifiers, where it was found that an intermediate flow rate of 0.9 mL/min was 2-5 times more stable in terms of number of injections prior to decrease in system plate height than the flow rates of 1.4 and 0.4 mL/min (the effect at slower flow rates was attributed to an increase contact time of sample Addition of ethylene with the organic mobile phase) (N10). glycol to organic mobile phases is recommended to prevent protein denaturation (N10).
RESTRICTED ACCESS MEDIA Overview. Restricted access medium is a general term for a packing material havin a hydrophobic interior covered by a hydrophilic barrier. !'he hydrophilic barrier allows passage of small molecules to the hydrophobic part of the stationary phase, while sterically preventing large molecules, such as proteins, from interacting with this part of the stationary phase. The to ic of restricted access media has N97,Nl50). There are several been recently reviewed s includin internal-surface reversed phase, semi ermesurface, stielded hydrophobic phase, and mixe: functional phase. These techniques have been compared (N98, N151).
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CLINICAL CHEMISTRY
Internal-Surface Reversed Phase (ISRP). ISRP was the first restricted access medium developed (in 1985) (N99). ISRP packin material consists of a high-performance silica support that fas a hydrophobic group attached to the surface inside the o m and hydrophilic groups attached to the surface outside tLe pores. It is unique1 prepared from silica containing immobilized polypeptd groups, which are selectively hydrolyzed from the support surface exterior to the pores with a proteolytic enzyme. Reviews describing this technique have been published (N13, N100, N101). The median pore size of ISRP supports is 52 A (N102), which has a size exclusionlimit for roteins of somewherebetween 15 000 and 25 000 Da (N101). $roteins are excluded from the pores by both a size exclusion and charge repulsion mechanisms (at least for the GFF support discussed below, in which negatively charged glycine groups are present at the pore entrance excludin negatively char ed proteins). Direct injection of the samde is done on the &RP column, with retention being controlledby the organic modifier content of the mobile phase. GFF Sup ort. (a) Description. The first ISRP sup ort immobilize&he pol eptide glycine-L-phenylalanine+ Eenylalanine (GFF)to tgsupport and formed hydrophilicggcine oups on the surface exterior to the pore by hydrolysis of the FF using the enzyme carbox eptidase A (N99). This is currently the most utilized R R P column owing to its commercialavailability (RegisChemical Co.). The retention mechanism of the GFF column is predominantly through r interaction with the phenyl groups of phen lalanine and secondarily through cation exchange with tKe negatively charged glycine groups (N103). The retention characteristics are similar to phenyl columns, which are less retentive toward hydrophobic compounds than C8 and CIScolumns (N99).An improved synthetic rocedure for makin GFF ackin material has doubletthe retention capabiyity of t i e GF! support in comparison to that produced by the former synthetic method (N104). (b) Performance. The longest column lifetime reported for GFF is over 2000 in'ections of 20 pL of serum sample, with the replacement of t i e guard column after 400 injections (N29), which is lower than the maximum column lifetimes reported for the precolumn techniques (see Table N-11). The efficiency of the new-generation GFF columns is 60000 plates/m (N104). Recovery studies of protein-bound drugs are few, with the following bein reported 100% recovery for lidocaine (65-77% prptein tound), 98% recovery for probenecid (83-9475 protein bound), and 100% recovery for cefpiramide (96% protein bound) (N103). This study found that the presence of 2.5% organic modifier was necessary to increase recovery (from 85% to 100% for cefpiramide) and improve efficiency (from a plate height of 350 to 140-190 pm) (N103). Serum proteins (99%) eluted from the column nonretained (N13). The nonretained peak was found to elute off the GFF column within 4 min while monitoring at 240300 nm, but 18 min for detection below 230 nm (1mL/min, 15-cm column) (N100). ( c )Advantages and Disadvantages. The major advantage of ISRP technique over the precolumn technique is that the ISRP technique has less sophisticated equipment needs, not requiring an extra pump, column switching apparatus, or computer control of events. There are several disadvantages to GFF ISRP technique in comparison to the precolumn technique. Detection is limited to drugs at or above the microgram per milliliter level (UV detection at 254 nm), due to the ISRP technique not havin the concentrating ability that is inherent in the precofumn techni ue, as well as resulting from interference of endogenous su%stances, which are minimized in the precolumn technique (N13). To address this issue, GFF ISRP columns have been used as precolumns to concentrate analyte rior to eluting analyte to a conventional column (N13,N99, 105-N107). However, there is no demonstrated advantage to using ISRP precolumns in lace of the reviously discussed reversed-phaseprecolumns. GFF I S d y l u m n has been used to determine a dopamine agonist in p asma at a detection limit of 1.5 ng/mL using electrochemical detection (N105). Another disadvan e to GFF ISRP is the limited flexibility in altering retention c aracteristics of the column. Protein precipitation limits organic modifier concentration (
