Trends in Chromatography

Within the scope of a number of emerging contaminant issues in environmental analysis, one area that has received a great deal of public interest has been the assessment of the role of pharmaceuticals and personal care products (PPCPs) as stressors and agents of change in ecosystems as well as their role in unplanned human exposure. The relationship between personal actions and the occurrence of PPCPs in the environment is clear-cut and comprehensible to the public. In this overview, we attempt to examine the separations aspect of the analytical approach to the vast array of potential analytes among this class of compounds. We also highlight the relationship between these compounds and endocrine disrupting compounds (EDCs) and between PPCPs and EDCs and the more traditional environmental analytes such as the persistent organic pollutants (POPs). Although the spectrum of chemical behavior extends from hydrophobic to hydrophilic, the current focus has shifted to moderately and highly polar analytes. Thus, emphasis on HPLC and LC/MS has grown and MS/MS has become a detection technique of choice with either electrospray ionization or atmospheric pressure chemical ionization. This contrasts markedly with the bench mark approach of capillary GC, GC/MS and electron ionization in traditional environmental analysis. The expansion of the analyte list has fostered new vigor in the development of environmental analytical chemistry, modernized the range of tools applied, and has revealed the need for awareness of the parallel developments in pharmaceutical analysis and biomedical analysis. We place particular emphasis on the separations that undergird successful analysis of PPCPs and the final separation/detection that provides the primary data upon which risk assessments and other determinations will ultimately be based. We suggest that the new emphasis on PPCPs has now defined a turning point in environmental analysis and set the stage for a significant new challenge that we briefly explore in this appraisal of the field.

Key words
Pharmaceuticals, separations, personal care products, gas chromatography, liquid chromatography, mass spectrometry, environmental analysis, effluent, surface water, capillary electrophoresis, laser-induced fluorescence, SPE, antibiotics, estrogens

Abbreviations
BSTFA: N,O-bis(trimethylsilyl)trifluoroacetamide; C18: octadecyl derivatized silica column packing; CE/LIF: capillary electrophoresis/laser-induced fluorescence; EC NIMS: electron capture negative ion mass spectrometry; EDCs: endocrine-disrupting compounds; EI: electron ionization; ESI: electrospray ionization; ECD: electron capture detector; DAD: diode array detector; GC/MS: gas chromatography/mass spectrometry; ELSD: evaporative light scattering detector; HPLC: high performance liquid chromatography; MT: migration time; PNAs: polynuclear aromatics; PPCPs: pharmaceuticals and personal care products; POP: persistent organic pollutants; RT: retention time.

INTRODUCTION

The determination of the occurrence and fate of pharmaceuticals and personal care products (PPCPs) in the environment is often considered an emerging area of environmental analysis.^{1, 2} In fact, the issue is not new. For example, the occurrence of PPCPs has been reported for some time with early papers by several authors3-10 for example, and an even earlier paper by Watts et al. ¹¹ What perhaps is meant is that interest in PPCPs has become widespread and engaged the attention of official government institutions such as the U.S. EPA and the U.S. FDA and similar agencies in other countries. Some of this impetus must be laid to the impact of the review article by Daughton and Ternes¹ and the availability of the web sites¹² devoted to this issue. The public has clearly identified with the potential effects of PPCPs and has understood the implications of personal actions in contributing to environmental contamination. This, in itself, is an important result that counters the impression that environmental problems result only from large corporations or chemical producers. The news media has amplified this interest and educational institutions have used the environmental occurrence of PPCPs as a vehicle for teaching environmental chemistry and science.

In this appraisal we attempt to outline and examine in more detail some of the major analytical approaches that are being used to determine PPCPs in the environment. Our emphasis is placed primarily on the relevant separations involved in the isolation, cleanup, and final separation/determination that produce the data needed for risk determination and other purposes. The sheer numbers of potential analytes and their diverse chemical properties have transformed the field of environmental analytical chemistry. By this we mean that the traditional emphasis on hydrophobic, bioaccumulated contaminants such as PCBs and polynuclear aromatics (PNAs) has given way to interest in extremely polar analytes such as those provided by many modern pharmaceuticals such as macrolide and fluoroquinolone antibiotics. The change in focus has been, in our opinion, extremely beneficial to a field that has lost some of its luster as a result of a rather stagnant analyte list and, in a sense, to the success of the methods developed in the past that are largely based on capillary GC/MS with a predominate EI approach. We should mention that polar analytes are certainly not unknown to historical environmental analysis and are clearly evident in certain pesticide classes such as a number of very polar herbicides. Thus, there are many U.S. EPA water methods involving such compounds, and such methods primarily use HPLC for the final separations step.

The innovation and energy and diversity of methods exhibited in fields such as clinical chemistry, pharmaceutical analysis, and biomedical analysis have been largely unshared in the environmental field until recently. In our opinion, the field has been slow to adapt new technologies and those that were new quite a while ago. For example, although electron capture negative ion mass spectrometry (EC NIMS) has been around for about 25 to 30 years, there is as yet no official U.S. EPA method utilizing this technique. This contrasts with its robust application in clinical chemical analysis. To be sure, there are GC/ECD methods in environmental analysis that ultimately involve gas phase negative ion chemistry. This observation carries over into many other techniques as well such as capillary electrophoresis/ laser-induced fluorescence (CE/LIF), LC/MS and LC/MS/MS that are common in clinical and other analyses but unfortunately lacking in official environmental methods.

Multidimensional separations have been by and large ignored by the environmental field, conceivably due to the added burden placed on analysis of large numbers of samples. The adoption of GC/GC/MS as a routine tool is nowhere to be found nor is there any implementation of the various multidimensional high resolution separations (e.g., LC/GC, GC/GC, and LC/CE). The adoption of improved apparent resolution in chromatography by way of fast mass spectral scanning in time-of-flight mass spectrometry (TOF-MS) seems to be slow to catch on. There is a startling lack of high resolution mass spectrometry (GC/HRMS) monitoring methods beyond dioxins and possibly PCBs despite the obvious power for the traditional analytes we have mentioned. One would think that such an approach would have use for applicable analytes at the very low part-per-trillion (ppt) levels that are involved. The strengthening of the specificity and selectivity of the detector is one of the chief strategies employed by chromatographers to lessen the burden on the separations that precede that determination. We suggest that a rather stagnant analyte list has had the effect of slowing the adoption of new technologies especially where they are developed to handle the challenges of new analytes requiring new methodologies.

The pursuit of PPCPs has therefore fostered greater emphasis on HPLC separations and, to a lesser extent, on other liquid separations such as CE. There now exits a parallel need for effective cleanups of polar analytes in the analysis of environmental samples that has often led to a different determinative step for many compounds. One approach is to use derivatization where practical and still rely on the power of GC/MS methods. Since many of these compounds are determined at ppt or sub-ppt levels in water or other matrices, the applicability of extended GC/MS methods is highly effective. In those instances where LC/MS is the logical and practical approach, the more limited separation power and greater complexity of liquid separations is keenly felt. Thus, the adoption of MS/MS approaches to PPCPs is common although we cannot recall a single U.S. EPA method in SW-846 involving GC/MS/MS to date (Method 521 involves CI and GC/MS/MS while Method 535Rev1.1 involves LC/MS/MS). Thus, we see the application of newer techniques common in pharmaceutical analysis and biomedical analysis finding their way into environmental analysis of PPCPs by way of necessity and because methods developed at this date reflect more clearly the current state of the art.

We suggest that the emphasis on PPCPs has now defined a turning point in environmental analysis and set the stage for a significant challenge. The separations now need to address a much more polar and diverse universe of analytes with the inherent difficulties of liquid separations versus capillary gas chromatographic separations. The paper cited above by Watts et al.¹¹ is representative of the change in emphasis and the role of improvements in technology in advancing the field. In Watts' paper, antibiotics were found in river water using HPLC and off-line field desorption (FD) MS. At the time, FDMS was one of the few techniques available for handling non-volatiles but was by no means universally adopted because of its sometimes arcane nature in the details of its application. The subsequent advent of LC/MS techniques such as APCI and ESI have now presented the field with practical tools to target non-volatiles on a widespread scale. However, the discovery of non-targeted and previously unknown non-volatiles remains a daunting challenge relative to the power of full scan GC/MS approaches to semi-volatiles and volatiles. We recall the contributions of GC/MS and EI in revealing many environmental issues of the past in the areas of persistent organic pollutants (POPs) and other analytes in the pursuit of unidentified peaks in sample extracts. The challenge of such peaks in LC/MS is equally important but of relatively more complexity due to apparent separations limitations, the limited availability of large databases of relevant mass spectra, the lack of a universal technique to obtain spectra rich in numerous correlations leading to the determination of organic structure, and difficulties in achieving accurate mass measurements relative to GC/MS. We also note that recent developments in the area of Homeland Security issues and analytical requirements in response to these developments will likely serve to reinforce the change in focus to separations of polar molecules and a more diverse analyte universe.

Source and occurrence
The source of PPCPs is primarily human (as well as domestic animal) use and activity including manufacture and disposal followed by transport via a sewage system, treatment, and wastewater effluent discharges into the environment.^{1, 13-17}  Thus, depending on compound loadings and efficiency of treatment, up to 90% or more of influent stream levels are removed before discharge resulting in 10-fold to 100-fold reductions compared to effluent.  Efficiencies of removal are highly compound dependent, however, and may be poor in some cases with the result that effluent and influent levels are comparable (in some cases effluent levels increase relative to influent due to such things as hydrolysis of conjugates).  Many PPCPs are present as metabolites or as environmental transformation/degradation products of the parent compounds.  Thus, clinical studies of pharmaceuticals are an important adjunct in identifying potential analytes.  Figure 1 summarizes some of the key issues and interrelations of this environmental research area.

Effects
The health effects of low levels of PPCPs in the aquatic environment are largely unknown. The concern is motivated because pharmaceuticals themselves are developed to effect a specific biological action in a target species. What is not clear is what biological response might be elicited from a non-target species either as a toxic effect or as a subtle sub-lethal effect in a number of areas such as behavior or reproduction that could prove deleterious over time.^{1, 2}  
Some examples of known effects include an EC50 value of 0.25 ppb on Daphnia magna for musk xylene reduction products.¹  Another is the effect of fluoxetine (Prozac) at 125 nmol on crustacean reproduction such as in fiddler crabs and fluvoxamine (Luvox) at nmol levels eliciting spawning in male mussels.¹  Issues such as synergistic effects and non-target species sensitivity are just beginning to be studied.  Pharmaceuticals are by design capable of potent actions on metabolic systems.  What is unclear is to what extent they affect other metabolic systems of non-target organisms subject to continuous exposure with cumulative effects possibly irreversibly affecting homeostasis.¹ Current research into effects includes work on antibiotics.¹⁸ and effluent.¹⁴

Risk
Since scarcely anything is known of effects or even of levels of PPCPs for most ecosystems, very little is known about the potential risks associated with their occurrence.  What is significant, however, is that their occurrence in the environment is often of the same order of magnitude as the persistent pesticides that are widely regulated.  It is not unreasonable to at least ask the questions of their occurrences, metabolic activities in nontarget organisms, and environmental sinks in order to begin to assess potential exposure and risk.²
A comprehensive risk assessment strategy has been proposed in several forums and some compounds have been evaluated.¹⁹.  A computational approach to initial estimates has taken the form of a structure activity relation (SAR) approach, and 2986 pharmaceuticals were evaluated by a quantitative structure activity relationship (QSAR) approach.²⁰

Separations/Determinations Organized by Compound Class

Introductory Remarks
We have selected several major compound classes of PPCPs.  This is not an exhaustive list but representative of a wide range of polarities that could be encountered.  We have included some examples of EDCs and what are almost traditional environmental analytes.  The intersection of PPCPs and EDCs is perhaps most apparent with the estrogenic compounds such as estradiol and ethynyl estradiol.  This class represents both naturally occurring and pharmaceutical representatives.  The polarity of this class we term polar hydrophobic, and it is decidedly more polar than familiar examples of EDCs such as PCBs and the other POPs. This is an interesting interfacial region where either HPLC or GC following derivatization can be pursued.  There are probably good arguments on either side of the approach issue, but our rule of thumb in environmental analysis is to use GC if possible because of its relative separations power, its reproducibility, and its relative ease of portability to other laboratories.

There is a practical reality to the adoption of methods by those charged with routine monitoring or facing large sample loads of diverse analytes.  Thus, the ability to dedicate a particular column and instrument to a diverse set of problems is an obvious advantage relative to sample throughput.  If variations are required depending on analyte, it would be better to have them occur prior to the final separation/determination so that the same overall instrument setup can be used with some changes in the details.  Thus, the selection of unique columns for each analysis is not readily embraced.  Likewise, adoption of multidimensional approaches will not be favored because, of necessity, more samples result from the fractionation and in many cases each will require concentration (and perhaps solvent exchange) since chromatographies are usually non-focusing (i.e., sample analyte is collected in a larger volume than that injected).  However, the application of multidimensional separations remains one of our most powerful tools for improving the selectivity of our separations.  Thus, there is a continuing resistance to change in methods as well as considerations of practicality and cost of implementation in the adoption of newer techniques.  Necessity is probably the overriding factor in the introduction of technological advances in performing critical analyses.  An additional stimulant may be the need for a preponderance of evidence to support regulatory enforcement.

Several comprehensive reviews have appeared that cover various aspects of PPCPs analysis including the review of Daughton and Ternes.¹  More recently, Ternes¹⁴ has reviewed aspects of the analytical chemistry of PPCPs, and Petrovic et al.²¹ have reviewed the role of LC/MS/MS in their analysis.  We are not aware that anyone has focused primarily on the separations aspect to date.
Before embarking on more detailed discussion of particular compounds, there are a few general comparisons we would like to make between the historical GC/MS EI approach and the newer LC/MS and LC/MS/MS approaches that are derived from our operational experience.  In some cases these differences are narrowing considerably with the introduction of new technology and the trend is that the techniques are approaching a par with each other in performance measures.

1. The LC/MS signal is usually significantly noisier.
2. LC/MS is less sensitive and has a higher detection limit than GC/MS.
3. LC/MS ionization has a strong dependence on surface activity and is subject to ion suppression whereas EI response is largely similar and with no suppression normally.
4. Absence of a response for a compound by LC/MS (i.e., one that responded as a standard) is not unequivocal evidence for the absence of the compound in the sample in contrast to most GC/MS EI work where response of a standard and the analyte in the sample are roughly equivalent .
5. Supplementary data providing alternative quantitation and assessing separations is often useful along side of LC/MS, e.g., DAD, fluorescence detection (FLD), and ELSD data.
6. LC has lesser peak capacity (total number of resolvable peaks) and apparent resolution than GC.
7. Reproducibility of RT for LC is 0.1 min versus 0.001 min for GC.
8. Ease of HRMS is greater in GC/MS than LC/MS.
9. Sample injection size is usually larger in LC than in GC (e.g., 50 µL versus 2 µL typical) but this can vary depending on format of the two.
10. Specificity of full scan data is greater in GC/MS (EI).

One issue that is particularly noteworthy for LC/MS is the ion suppression phenomenon and the concern for quantitative accuracy as a result of co-elutions.  Generally, assuming a valid sample work up, the absence of a response for a chromatographable compound in a sample by GC/MS is a valid basis for concluding its absence, especially when quality control (QC) is present in the form of an internal standard that validates instrument performance.  This may not be the case for LC/MS where the presence of excess salt or a surfactant, for example, could completely mask underlying compounds present.  Thus, the addition of other detection techniques such as the diode array detector (DAD) or the evaporative light scattering detector (ELSD) are useful adjuncts in assessing cleanup, sample quantitation, quality of chromatography, and presence of coelution.  An additional useful technique is to include at least one matrix spike with the sample set that validates expected response for the analyte/matrix pair.  Because of sample variability in environmental analysis, it is risky to assume that there will be typical behavior or constant matrix constituents.
Another factor to consider is that use of highly selective detection techniques results in a paucity of knowledge about the sample and other components in it. The monitoring of short retention time windows of specific LC/MS/MS transitions affords an extremely limited view of the overall sample responses obtained. Even operating in a full scan mode in LC/MS may provide a limited amount of information about sample constituents as a consequence of "soft" ionization and ion suppression effects. Thus, it is possible to be unaware of co-elutions that might affect ionization or of non-target analytes that are potentially important contaminants.

Separation and detection of the PPCPs
Table 1 provides a brief summary of some of the kinds of compounds under consideration in the area of PPCPs and to a limited extent the intersection with EDCs.  The table serves to provide a perspective on the chemical diversity and some measure of the polarity of the analytes along with a synopsis of methods that have been used in their analysis.  Our overview here will reflect an emphasis on the handling of the polar hydrophobics and very polar analytes.  Along the way we will point out some technologies that have not been applied or used sparingly that could be applicable to these analyses.  We will emphasize mainly changes in SPE, cleanup, and new phases for HPLC.  Several kinds of PPCPs are available as substances of commerce, and pharmaceuticals in particular fall into two broad categories: prescription and over the counter drugs.  A third category consisting of illicit drugs may also need to be considered.  Pharmaceuticals may be classified on the basis of the mode of action or symptom that they treat.  Thus, we have examples from antibiotics, analgesics, anti-depressants, anti-hypertensive, lipid regulators, hormones, stimulants, and disinfectants as representative classes.  Briefly we have listed the methodologies for separation and detection of PPCPs including HPLC-MS/MS and GC-MS/MS with samples drawn from different environmental matrices.  Details of these methods are individually referenced.^22-38  Ternes has provided schema for acidic, neutral, and basic analytes that can be useful in approaching each class for analysis.¹³
We have provided some example structures to go along with the discussion.  Chromatographers look at the chemical structure of the analyte in order to assess what types of separations might be appropriate.  The physical and chemical properties of the substances are also valuable, especially solubility, pK_a, pK_ow, and melting point or boiling point.

Acidic pharmaceuticals
Acidic drugs are an important group of pharmaceuticals that contain carboxylic acid moieties and usually one or more hydroxy groups.  All these compounds can be nearly quantitatively enriched on C18 cartridges at pH 2-3 from an aqueous matrix.¹³   At the acidic pH no ionic functional groups are present since the hydroxyl and carboxylic acid moieties are unionized.  Analytical procedures consisted of SPE using RP-C18 cartridge, followed by methylation of carboxylic groups with diazomethane, acetylation of phenolic hydroxyl groups with acetic anhydride/triethylamine (1:1, v/v), and determination by GC/MS. It would also be possible to silylate the hydroxy and carboxylic functions with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)³⁹ before GC/MS analysis. Typical examples with structure of acidic drugs are given that belong to several groups of pharmaceuticals.

Gemfibrozil, CAS: 25812-30-0, molecular mass: 253.33 (lipid regulator).

Acetominophen, CAS: 103-90-2, molecular mass: 151.16 (analgesic).

Salicylic acid, CAS: 69-72-7, molecular mass: 138.12 (antiseptic).

Phenol, CAS: 108-95-2, molecular mass: 94.11 (disinfectant).

Application of newer phases for SPE such as the Oasis HLB^® sorbents can be made with less concern about drying out the sorbent and with faster initial conditioning.¹⁴ Alternatives to diazomethane methylation consist of many different methylating reagents including trimethylsilyldiazomethane.⁴⁰  Such an approach was used for GC/MS after screening by CE/LIF.⁴¹  Fig. 2 illustrates what effluent affords for acidics using free zone electrophoresis with laser-induced fluorescence at 244 nm excitation.  Salicylic acid is noted at migration time 6.65 min with the electroosmotic flow peak at 4.26 min and internal standard at 7.8 min.  Obviously, free zone electrophoresis is inadequate to resolve all of the fluorescent compounds present based on ion mobility alone.  CE is particularly powerful when coupled with HPLC in a multidimensional format where fractions are subjected to CE separation.
Acids can also be determined by LC/MS in either the positive or negative ESI.  Farre et al. used a C18 LiChrospher column with negative ESI for acidics.⁴²  Fig. 3 shows a chromatogram that includes acidics for LC/MS/ESI(-) in a sample of river water.

Neutral pharmaceuticals
Neutral pharmaceuticals are a group of drugs from several medicinal classes which contain no acidic functional groups but may contain bases.  These can be extracted from water at neutral pH (6-7.5) by C18 cartridge, and can be analyzed by GC/MS without derivatization or by LC/MS.  Examples of a few neutral drugs from different medicinal groups are given below.

Fluoxetine, CAS: 54910-89-3, molecular mass: 309.33 (antidepressant).

Carbamazepine, CAS: 298-46-4, molecular mass: 236.27 (antiepileptic).

Etofibrate, CAS: 31637-97-5, molecular mass: 363.79 (lipid regulator)

Ifosfamide, CAS: 3778-73-2, molecular mass: 261.09 (antineoplastics).

An example from this class is carbamazepine which is among the most widely found drugs in the environment.¹

Any drugs from this class or other classes that are strongly basic or can act as a base under aqueous conditions may be extracted using cation exchange approaches.⁴³  In such cases these drugs may be highly amenable to LC/MS ESI (+) determination. 

b-blockers and b₂-sympathomimetics

-blockers and ₂-sympathomimetics pharmaceuticals are generally recommended as anti-hypertensive drugs.  Both classes of compounds contain secondary aminoethanol and one or more hydroxy groups in their structure, and, as a result, their polarity is relatively high.  For GC analysis of the drugs, efficient derivatization is important.  Sample preparation from water includes SPE, derivatization by silylation of the hydroxyl groups and trifluoroacetylation of the secondary amino moieties.  LC-ESI(+)/MS/MS is also used with a gradient of water/acetonitrile containing ammonium formate buffer using a RP-C18 analytical column.³¹  Structures and other information of typical -blockers and ₂-sympathomimetics are given below.

Propranolol, CAS: 525-66-6, molecular mass: 259.34 (b-blockers).

Nadolol, CAS: 42200-33-9, molecular mass: 309.4(b-blockers)

Fenoterol, CAS: 13392-18-2, molecular mass: 303.35(b-sympathomimetics).

Salbutamol CAS: 18559-94-9, molecular mass: 239.31 (b-sympathomimetics).

Basic compounds are usually problematic for HPLC and a great deal of work has gone into improving columns for use with bases.  Usually, the pH of separation is around 3 which has the effect of suppressing the ionization of silanol groups remaining on the silica after all phase treatments.  These silanol groups generally lead to undesirable ion exchange interactions resulting in peak broadening or tailing.  The alternative use of ion-pairing chromatography has been reported.⁴²

There has been some discussion of the relative merits and retention characteristics of the usual C18 columns versus polar embedded or end-capped packings.  The polar embedded columns were developed to improve the chromatography of basic compounds and to allow 100% aqueous mobile phase leading to better retention of polar analytes.  The selectivity is changed from that of C18 as well. ^{44, 45}  A different strategy using difunctional bonding resulted in the dC18 columns such as Atlantis^®.  In this case the retention of polar molecules is increased, normal retention similar to C18 is observed for less polar analytes, and good aqueous compatibility is also achieved.⁴⁶ 

Hormones and steroids
A great deal of focus has been put on EDCs many of which are estrogenic in action but the class includes androgenic compounds as well.  These compounds contain the characteristic steroidal structure with various hydroxy groups. Estrogens possess a higher lipophilicity (log P_ow 3.5-4.6) compared to other human pharmaceuticals, and their excreted quantities are extremely small.  The appropriate analytical method with highly sensitive detection tool is required for finding these compounds in environmental samples.  The natural and synthetic estrogens can be analyzed simultaneously because of their similar physico-chemical properties.  Environmental water samples could be extracted with an SPE cartridge using Lichrolut EN^® and RP-C18 at pH 3.  Cleanup was by silica gel followed by derivatization with N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA)/trimethylsilylimidazole (TMSI)/dithioerythritol (DTE) (1000:2:2) and detection by GC-ion trap-MS/MS.³²  LC-ESI(-)/MS with SIM was employed for estrogens analysis using a C18 column with acetonitrile/water eluent.³³ Examples and chemical structures of estrogens are given below.

17b-estradiol, CAS: 50-28-2, molecular mass: 272.38.

Progesterone, CAS: 57-83-0, molecular mass: 314.46

Estriol, CAS: 50-27-1, molecular mass: 288.38

Coprostanol, CAS: 360-68-9, molecular mass: 388.67

Antibiotics

Antibiotics are frequently used drugs in both human and veterinary applications. Antibiotics cover a wide range of drugs including macrolides, tetracyclines, sulfonamides, quinolones and fluoroquinolones, and penicillins.  Each group possesses chemical structures that are different from other groups.  A number of functional groups such as –OH, –COOH, =C=O, =S=O, =NH or substituted groups may be present in one antibiotic so that the SPE isolation of antibiotics from environmental samples could be applicable in the pH range of 3 to 7.  Several SPE cartridges and phases, e.g. Oasis^® HPLC, RP-C18, and LiChrolute EN^®, have been employed to extract samples.³⁸  The separation and detection were made by HPLC-ESI-MS/MS using a C18 column with a recommendation of guard column use prior to analytical column.  Typical examples of different medicinal classes of antibiotics are shown.

Erythromycin, CAS: 114-07-8, molecular mass: 733.93 (Macrolide group).

Doxycycline, CAS: 564-25-0, molecular mass: 444.43 (Tetracycline group).

Ciprofloxacin, CAS: 85721-33-1, molecular mass: 331.34 (quinolones and fluoroquinolones group).

Sulfamethoxine, CAS: 651-06-9, molecular mass: 280.30 (Sulfonamides group).

Cloxacillin, CAS: 61-72-3, molecular mass: 435.88 (Penicillin group)

In Fig. 5⁴⁸ we show the peak width for amoxicillin (RT=4.29 min) using a separation for several antibiotics and other compounds.  The peak width at half height is between 4 and 5 sec so that reasonably good peak shape and efficiency are obtainable by LC/MS but typical peaks often exhibit greater widths at half height of 9 sec or more.  The retention times of azithromycin, urobilin, and clarithromycin were (15.47, 20.18, and 25.0 min respectively). ⁴⁸

In Fig. 6⁴⁸ the application of multidimensional chromatography for determining fluoroquinolones is illustrated; the cleanup step is a semi-preparatory HPLC fractionation  of the sludge extract after partitioning in water/methanol versus hexane.  The fluoroquinolones are visible as a small peak (UV-254 nm).  This fractionation is followed by an analytical separation (but incomplete resolution all the compounds, at least three fluoroquinolones about 28 ppb) in the determinative step using HPLC/FLD in Fig. 7⁴⁸; a much better separation of fluoroquinolones is shown from the literature in

Fig. 8.^{49, 43}  Gobel et al. have also determined antibiotics in sludge.⁵⁰  

Antibiotics can be problematic for analysis because of decomposition (e.g., amoxicillin prepared as a solution for medicinal purposes is only recommended for two weeks use stored in the refrigerator).  It is challenging to achieve separations of all members of a given antibiotic class and, in addition, separate the other classes  as well.  The separations are usually carried out under acidic conditions (pH 3), with a predominantly aqueous mobile phase and developed with a very shallow gradient in the organic solvent for elution of the compounds.

Personal care products

The relatively inexpensive synthetic nitro musks such as musk xylene are frequently used as fragrance materials in formulation of personal care products, and commercial toiletries.  Nitro musks enter city sewage systems and aquatic ecosystems where they may potentially bioconcentrate in the tissues of aquatic organisms, or they may deposit in the sediments since their biodegradation rate is slow and their lipophilicity is high. Varian Abselut Nexus^® SPE cartridges extract both polar and non-polar compounds from aqueous environmental samples. Desorption of SPE with hexane, followed by silica gel cleanup helped to detect the nitro musks and their metabolites by GC-MS using HP-5^® MS or DB-5^® MS capillary column (30 m x 0.25 mm id x 0.25 um film thickness) without derivatization.³⁵  Chemical structures of mono- and polycyclic musks are shown.

Musk xylene, CAS: 81-15-2, molecular mass: 297.26

Galaxolide, CAS: 1222-05-5, molecular mass: 258.40

Musk moskene, CAS: 116-66-5, molecular mass: 278.30

Fig. 9 illustrates the enhanced selectivity of ECNIMS for amino musk metabolites isolated from fish hemoglobin.⁵¹  EI mass spectra were also obtained during these analyses enabling a fuller assessment of components in the extract. ⁵¹ 

As described earlier, this is an overview of the trends that are apparent where we have described some of the separations used for PPCPs.  Table 1 summarizes a representative list of PPCPs with general use, extraction method, matrices, separation techniques, and detection mode including relevant references.   The review of Ternes¹³ is a good starting point for locating an approach to a particular class of compounds, although the focus is almost entirely on an aqueous matrix.  We have tried to offer some sense of the chromatography achieved, primarily with illustrations from real samples.  Many works in the literature fail to show examples of the chromatography achieved with either real samples or standards.

Current and Future Trends

To date the current  matrices have been mostly aqueous leaving a significant amount of work with solid matrices such as soil, sediment, food, biota, and biosolids.  The role of ion exchange SPE is now more important since a number of acids and bases have been added to the analyte lists.  Mixed mode or mixed bed SPE is sometimes used but the advantage of this over separate, optimized cartridges in series that yield separate fractions is not entirely clear to us.  One obvious “hole” in the liquid-liquid extraction separations of acids and base/neutrals from the classic U.S. EPA methodology is presented by the amphoteric compounds such as the fluoroquinolone antibiotics.  They will always be ionized under the usual conditions of the acid and base/neutral partitionings and therefore remain in the aqueous fraction.  This possibility has led to an essential application of cation exchange for isolation/cleanup of the bases.  Caution is noted when using strong cation exchange because large organic bases may be difficult to elute.  An alternative is to use the weak cation ion exchange based on a carboxylate group of the exchanger and control the pH for retention (e.g., pH 5 - 7) followed by elution at pH 2-3 for example (suppressing the ionization of the carboxylic acid group).

Development of newer SPE such as the Nexus^® and Oasis^® cartridges appear to have strengthened the chemistry for isolation steps⁵².  Major problems with SPE techniques have involved variability in recoveries, handling particulate matter, and drying out of the bed.  Likewise, the application of polar-modified C18 and increased polar retention phases should see continued growth and applications.  According to one description, the polar modified phases improve the column performance at very high water content of the mobile phase whereas a phase such as Atlantis improves polar molecule retention and therefore for preparative work allows convenient concentration because of higher organic content. 

Applications of ultra performance LC (very small particles and very high pressures), monolithic columns, and zirconium columns seem lacking so far in the environmental analyses.  We predict that the HPLC separations will continue to improve in peak capacity as smaller particle sizes become more routine (e.g., 1.7 µm) and higher pressures become actively used in PPCPs analysis.  This will both shorten run times and lead to narrower peaks, and thus place the liquid separations on a par with capillary GC separations.

At present we are able to separate at most perhaps 100 to 200 compounds with a single column.  If we need to separate 10,000 potential analytes, then two-dimensional separations appear to be one alternative.  The second alternative has been to use a more selective detection, and mass spectrometry has played a major role with two complementary techniques each of which is based on “separations” in the mass dimension.  One is to use high resolution mass spectrometry to resolve closely lying mass to charge ratios (ions) and the other is to use MS/MS and produce unique product ions that can distinguish among analytes.  The combination of HRMS (i.e., on the initial ion beam) with MS/MS has been rarely used.  In contrast, new technology implementing accurate masses and moderate resolution with MS/MS has become more commonplace with regard to the product ion dimension.  This latter trend is probably inevitable and a necessity for liquid separations.  One pressing need is to produce full scan mass spectra that are rich in fragment ions for structural elucidation at the detection limits of interest.  At present “soft ionization” and MS/MS often do not yield enough useful fragmentation to allow structural elucidation in many cases. 

Capillary LC and capillary electrochromatography (CEC) appear to have generated little interest among workers involved with PPCPs analysis. Generally, capillary format separations such as capillary LC, CEC, and CE have not offered particular advantages to environmental analysis because of the robust sample size usually available from the environment.  Where sample size is less than a gram such as in biopsy sampling of biota, there may be a niche role for capillary liquid separations to play.  Issues of detection limits (absorption versus laser-induced fluorescence detection) and ruggedness in both the sense of sample loads and laboratory intercomparability seem to persist relative to capillary liquid separations. 

Notice:  The views expressed here are those of the individual authors and do not necessarily reflect the views and policies of the U.S. Environmental Protection Agency (EPA).  Scientists in EPA’s Office of Research and Development (ORD) have prepared the EPA contributions and those contributions have been peer and administratively reviewed and approved for publication.  Mention of trade names or commercial products does not constitute endorsement or recommendation by EPA for use.

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Figures captions

Figure 1.  Interrelationships concerned with the introduction of PPCPs into the environment.  Adapted from ref 12 and used by permission of C. G. Daughton.

Figure 2.  Screening for fluorescent acidics in an extract of effluent by CE/LIF.  Conditions:  Capillary was  58 cm long with window 8 cm from end and 0.075 mm ID; running buffer was 20 mM borate buffer and separation voltage was 18 kV; excitation wavelength 244 nm.  Salicylic acid MT=6.65 min. Adapted from ref 41.

Figure 3.  Analytical separation of acidic drugs in water by LC/MS/ESI(-).  Conditions:  Lichrospher 100 RP-18 column using acetonitrile (A) and water (B) both acidified to pH 2.0 by formic acid following a program of  2 min 30% B initial, linear gradient to 40% B in 5 min, isocratic 2 min, linear gradient to 60% B in 11 min and held there for 10 min.  Identifications by RT: 8.2 min, salicylic acid; 16.9 min, naproxen; 22.6 min, diclophenac--Na; 23.3 min, ibuprofen; 28.0 min, gemfibrozil.  Adapted from ref. 42.

Figure 4.  Estradiol (m/z 416), RT=14.11 min (internal std 15.71 min) determination by GC/MS (EI) after silylation in an extract of sewage effluent using SPE silica cleanup (hexane, methylene chloride/hexane, methylene chloride, acetone fractionation) followed by a preparative HPLC-PGC cleanup step.   Conditions:  Agilent 5973 GC/MSD operated in the EI mode,  J&W DB5MS column (40 m long, 0.18 mm i.d., 0.18 µm film, 105-300°C @ 20 deg/min).  HPLC-PGC conditions:  Hypersil 5µm HyperCarb (porous graphitic carbon with a hexane, toluene, and acetonitrile gradient).  Adapted from ref 47.

Figure 5.  Analytical separation of standards of several PPCPs including macrolide and β-lactam antibiotics using LC/MS/MS:  Peak width of amoxicillin. Conditions:  Atlantis dC18, 3 µm packing, 150 X 2 mm id column, 0.1 mL/min flow; initial 30/70 (82:18 methanol:acetonitrile)/(water, .5% formic acid) with linear gradient to 90/10 at 24 min and held until 40 min; MS/MS of m/z 366 of amoxicillin scanning product ions from m/z 100-400.  Adapted from ref 48.

Figure 6.  Semi-preparative separation of  fluoroquinolones from extract of sludge following a partition between aqueous methanol and hexane.  Conditions:  Waters Atlantis dC18 (5µm packing in 100 X 10 mm id column with 5 mL/min flow), initial 85/15 water/acetonitrile for 5 min then 20 min duration linear gradient to 100% acetonitrile, 0.1 % formic acid in both solvents, LabAlliance HPLC 2500 pump (SSI) with JMST VUV-14 detector at 254 nm, laboratory-built data system using Symmetric Research ADC (basic acquisition/drivers software) with privately developed software for additional acquisition/processing.  Adapted from ref 48.

Figure 7.  Analytical determination of fluoroquinolones from sludge after preparative HPLC.  Conditions:  Phenomenex Luna C18(2) (5µm packing, 250 X 2 mm id column, 0.250 mL/min flow), initial 90/10 water/methanol for 5 min followed by 25 min duration linear gradient to 100% methanol,  0.1 % formic acid in both solvents, Beckman System Gold (126 pump) with 32Karat data system and diode array detector (168) followed by a LabAlliance Ultrafluor (LC305) fluorimeter (Linear Instruments) interfaced to an Agilent ChemStation data system for the transition ex278 em450 nm fluorescence for fluoroquinolones and ex278 em356 for internal standard nalidixic acid. Adapted from ref 48.

Figure 8.  Separation of fluoroquinolones adapted from.¹⁷ Conditions: Supelco Discovery RP-AmideC16 5µm packing, 250 X 3mm (with precolumn 20 X 3 mm), solvent A 25 mM H₃PO₄ water (pH 2.4) and solvent B acetonitrile; linear gradient initial 5% B to 7% B in 17 min followed by 5 min isocratic followed by 13 min linear gradient to 17% B.  Referenced from ref 43.
Figure 9.  Selectivity of capillary GC/negative ion electron capture MS for musk metabolites in fish hemoglobin (m/z 267 of 4-aminomusk xylene) at RT=25.7 min.  Conditions:  Agilent 5973 GC/MSD operated in the negative ion mode,  methane reagent gas, J&W DB5MS column (40 m long, 0.18 mm i.d., 0.18 µm film, 60-150°C @ 10 deg/min, 150-250°C @ 8 deg/min, 250-300°C @ 10 deg/min).  Adapted from ref 51.

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