Chromatographic analysis of bisphosphonates in the past was conducted basically on reversed-phase liquid chromatography (RP-HPLC) and ion-exchange chromatography . Gas chromatography (GC) and recently even capillary electrophoresis have also been employed. For bioanalysis, a fundamental role in bisphosphonates assays is the pre-treatment of the sample. Derivatization is used in GC to form volatile compounds. For RPLC we can modify the bisphosphonate side chain by derivatization with a chemical reaction to yield compound with advantageous spectroscopic properties especially with fluorescence detection or complexate the phosphonate groups, or the phosphate after decomposition of the analyte to form a coloured complex. The first methods in bisphosphonate analysis, developed for etidronate [(1-hydroxyethylidene)-bisphosphonate] measurement in biological media, were non-chromatographic and were based on either titration with thorium diaminocyclohexanetetraacetate  for faeces and urine samples or on detection of phosphate as a triethylamine-phosphomolybdate complex after decomposition of the bisphosphonate  for urine and plasma samples respectively. These methods were very laborious but sensitive enough to measure etidronate concentrations in these biological media during or shortly after the parenteral  or oral  administration of the drug. Bioanalytical methods, developed later for several bisphosphonates of newer generations, are mostly based on chromatography. The introduction of chromatographic bisphosphonate assays was possible because of the tempestuous development of separation science since the first clinical experiments with bisphosphonates were started. However, there may also be another reason that both non-chromatographic methods are no longer applied: the newer bisphosphonates are more potent and therefore administered in lower doses   , increasing the demands on the sensitivity of the analysis. In addition to bioanalytical methods, bisphosphonate assays have also been reported in other working areas. Pharmaceutical analysis is an important field where several chromatographic methods have been developed for the quality control of pharmaceutical preparations such as tablets, injection concentrates and bone scintigraphy kits. However, analytical research on bisphosphonates, specifically methylenebisphosphonate and etidronate, was initiated in the early 1970s because of their application as complexation agents in washing powders. Chromatographic procedures usually comprise the following steps: sampling, storage, sample pre-treatment, analytical separation, detection, registration and interpretation. The most important steps are sample pre-treatment, analytical separation and detection.
Mostly, a chromatographic separation has to be preceded by one or more pre-treatment steps, especially for complex matrices. This may be done for several reasons:
1) A sample clean-up in order to remove endogenous components that disturb the analytical separation, for example: proteins, lipids, salts or coeluting components.
2) Concentration of the analyte to obtain a higher sensitivity; this is only possible if a sufficient sample is available.
3) Changing the physical properties of the analyte, for example by derivatization or complexation, to obtain an analyte suitable for a specific analytical method or to improve the detectability. Sample pre-treatment methods applied frequently in bisphosphonate assays are discussed in the next sections.
Most bioanalytical reports deal with the analysis of liquid biological samples such as urine, serum and plasma; however, a few methods regarding the analysis of solid samples, faeces and bone, have also been published. These solid samples have to be dissolved prior to further quantitative analysis. Faeces were refluxed in concentrated hydrochloric acid , bone was heated at 50 °C for 2 hours in concentrated hydrochloric acid   or dissolved in diluted hydrochloric acid after grinding and sifting it into small particles (<20 µm) .
Denaturation of proteins by precipitation is, in general, a well known method in the analysis of serum or plasma. For bisphosphonates, this method has also been applied to urine samples by Chester et al. . It is a fast and simple method compared to enzymatic methods  and, in contrast to ultracentrifugation, protein binding of the analyte can be interrupted, depending on the type of analyte, protein binding and precipitation method. The average protein bound fraction in human plasma in vitro varies between 25 and 90% for the five commercially available bisphosphonates . Because of its simplicity and the fact that it allows interruption of protein binding, protein precipitation is the denaturation method of first choice for the determination of the total concentration of a bisphosphonate in plasma or serum. Popular precipitation agents are perchloric acid, acetonitrile, trichloroacetic acid (TCA), and tungstic acid because of their efficient precipitation . TCA is the strongest precipitation agent: with 0.02% (w/v) in plasma, more than 99% of the proteins can be precipitated . Protein precipitation in bisphosphonate analysis has been performed with perchloric acid  and TCA       .
Calcium precipitation is a method by which bisphosphonates can be precipitated from an aqueous matrix. It is a treatment with an interesting specificity for this type of analyte, leaving most salts and organic species in solution. The bisphosphonates are precipitated as calcium complexes, together with calcium phosphate, by calcium addition, followed by alkalifying the solution. This is a process, analogous to the binding of bisphosphonates to the hydroxyapatite Ca10[PO4]6[OH]2 bone matrix  . The affinity of bisphosphonates to calcium hydroxyapatite and calcium hydroxide was applied in a bisphosphonate assay for etidronate in faeces and urine, respectively . Calcium precipitation with phosphate was developed into an adequate analytical tool, performed as a co-precipitation of the bisphosphonate with calcium phosphate, by Bisaz et al.  for etidronate. Later, several other bisphosphonates were isolated from biological matrices by this method, followed by liquid chromatographic (LC) analysis. Since the introduction of the co-precipitationmethod with calcium , several modifications and variations have been introduced. In general, the following procedure has been followed: the addition of phosphate, or occasionally pyrophosphate, to the aqueous phase is optional for urine or dissolved bone; however, for assays in plasma or serum phosphate addition is necessary to form sufficient precipitate. Calcium ions are added, often as calcium chloride, and a precipitate is formed by adding sodium hydroxide in a constant amount or until a specific pH is reached. A pellet is formed by centrifugation and the
supernatant is rejected. The pellet can be cleaned up by rinsing with water or by suspending it in water, water/acetonitrile or a sodium hydroxide solution, again followed by centrifugation and rejection of the liquid phase. After redissolving in hydrochloric acid, the precipitation procedure can be repeated. After the final precipitation the pellet can be redissolved in a solution of hydrochloric acid, ethylenediaminotetraacetic acid (EDTA), acetic acid, phosphoric acid or a mixture of these acids. The solution obtained can be injected directly into a LC system    or may have to be treated further          before final separation and quantification.
The multiple ionic character of bisphosphonates, extremely acidic conditions excluded, should make ion-exchange treatment a suitable preparation technique. Classical cation-exchange resins have been applied for converting the bisphosphonate into the acid form before silylation (see next section) , and for redissolving the residue after precipitation with hydroxyapatite . Such a resin was also applied in combination with EDTA to remove calcium ions from a dissolved calcium precipitate . In order to clean up a urine sample and to obtain the acid form of the bisphosphonate prior to silylation, an anion-exchange resin packed in disposable extraction columns can be employed for solid-phase extraction (SPE) . The analyte clodronate [(dichloromethylene) bisphosphonate] was eluted from the column with 2 M hydrochloric acid. The use of such columns makes the solid phase easier to handle and the procedure easier to automate. The solid phase in the column can also be based on silica. Diethylamine or quaternary ammonium modified silica columns were used for the redissolved calcium phosphate precipitate, primarily for the removal of calcium ions. The analyte alendronate [(4-amino-1 hydroxybutylidene)bisphosphonate] was eluted with the strong eluting citrate anion from the diethylamine column  , pamidronate was eluted with diluted nitric acid from the quaternary ammonium column  . After a calcium precipitation procedure, the calcium ions can also be removed by applying cation-exchange based SPE, retaining the calcium ions ; in this specific assay, olpadronate [(3-dimethylamino-1-hydroxypropylidene)bisphosphonate] could directly be collected during the loading of the sample on the column. Another possible molecular moiety for the application of SPE on bisphosphonates is the hydrophobic side chain of newer bisphosphonates. When this hydrophobic moiety is large enough to compete with the polar character of the basic bisphosphonate skeleton, the analyte can be retained on a more common octadecyl modified silica phase (C18). This has been performed in combination with calcium precipitation by Usui et al. for the determination of the bisphosphonates incadronate [((cycloheptylamino)methylene) bisphosphonate]  and YM-529 [(1-hydroxy-2-(imidazo[1,2-a]-pyridyl)ethylidene)bisphosphonate] . Chester  treated a urine sample, containing clodronate (which lacks hydrophobic properties), with C18-SPE columns; unfortunately the details of this procedure were not reported.
Derivatization, when applied in chromatographic analysis, comprises a chemical modification of the analyte in order to facilitate the application of a specific separation mechanism, to enhance the detectability and/or to improve the selectivity or specificity of the separation. For gas chromatographic (GC) analysis of bisphosphonates the first aim of these three will always be the most important since bisphosphonates are not volatile. In addition, it will always be advantageous to shield the polar groups of an analyte before GC injection; symmetric peaks will be easier to obtain. For LC bioanalysis of bisphosphonates, enhancing the detectability will often be the most important objective of a derivatization reaction; most bisphosphonates do not possess any moiety which facilitates a sensitive detection with common LC detectors. In general, most derivatization reactions are based on the replacement of an active hydrogen with an alkyl group (alkylation) or the exchange of nucleophilic groups (acylation) . In bisphosphonates, the four active hydrogens of both phosphonic acid groups should be replaced; this is a pre-requisite for volatilization of the analyte prior to GC. Active hydrogens of the different side groups, if present, may also be used for derivatization; hydroxy and amine groups particularly are frequently present Table I. Reported derivatizing agents for the GC analysis of bisphosphonates are N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), reacting with the phosphonic acid groups of clodronate  or the phosphonic acid groups and hydroxy group of etidronate , and the combination of isobutylchloroformate, reacting with the primary amine of pamidronate [(3-amino-1 hydroxypropylidene)bisphosphonate], followed by alkylation of the phosphonic acid groups with diazomethane  . Alternative derivatization procedures, applied to aminoalkylphosphonic acids, have been performed with a mixture of BSTFA and trimethylchlorosilane , a mixture of trifluoroacetic acid and trifluoroacetic anhydride, followed by a methylation with diazomethane , N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide  or a mixture of a fluorinated anhydride, trifluoroethanol or pentafluoropropanol, with a perfluorinated alcohol, trifluoroacetic anhydride, heptafluorobutyric anhydride or 2,2,3,3,4,4,4-heptafluoro-1-butanol   . Two types of derivatization reactions for the LC analysis of bisphosphonates have been described. One type of reaction involves the introduction of fluorogenic labels, coupled to the amino group of several,bisphosphonates. Pamidronate with fluorescamine   and different isothiocyanates     and alendronate with 2,3-naphthalene dicarboxyaldehyde (NDA)   , (9 fluorenylmethyl)chloroformate  and o-phthalaldehyde with mercaptoethanol  have been investigated. Recently, the first bioanalytical assay has been developed for olpadronate, a bisphosphonate with a tertiary amino group, based on a derivatization with (9-fluorenylmethyl) chloroformate . The NDA and 1-naphthylisothiocyanate   derivatization have proven to be the most successful with respect to a high sensitivity for bisphosphonates with a primary amino group. The NDA derivatization was performed in-line and post-column, the others were carried out off-line and pre-column. All the derivatives were separated by reversed-phase liquid chromatography (RPLC) and detected according to the corresponding specific detection property of the label. The second type of derivatization reaction is not based on a common organic synthetic reaction but on complexation, mostly post-column and in-line after ion-exchange chromatography (IEC). Metal ion-bisphosphonate complexes, suited for spectrophotometric detection, were created with three different ions. Aluminium(III) ions, added post-column as a morin complex to facilitate indirect fluorescence detection, were employed by Meek and Pietrzyk  and iron(III) ions were used by Fitchett and Woodruff , followed by ultraviolet (UV) absorbance detection at 330 nm. Copper(II) complexes of bisphosphonates were formed in-line by adding a copper(II) salt to the buffer for capillary zone electrophoresis (CZE)  or to the eluent for IEC ; the complex was detected with UV absorbance at 240 or 245 nm respectively. Virtanen et al.   formed a thorium-EDTA-bisphosphonate complex after post-column addition of a thorium-EDTA-xylenol orange complex for indirect spectrophotometric detection. Another possibility is a twostep reaction where the bisphosphonate is decomposed, post-column, into o-phosphate by added persulphate, at elevated temperature. This phosphate is then complexed with molybdate and the formed complex is reduced by the simultaneously added ascorbate   or vanadate  as a reducing agent. When ascorbate is employed, the well known complex molybdenum blue  is formed. The coloured reduced complexes formed by both reducing agents are detected spectrophotometrically. The advantage of these complex forming reactions is the applicability, in theory, to all bisphosphonates. However, fluorescent derivatives are in general detectable at lower levels in LC compared to the metal ion-bisphosphonate complexes detected spectrophotometrically. In addition to the fluorescent labels for pamidronate and alendronate already mentioned, several other labels may be chosen for the derivatization of primary (and often also secondary) amino groups into a fluorescent derivative . Hydroxy groups are present in most potent calciumregulating bisphosphonates and represent a chemical moiety which may be derivatized with nitriles . However, an ideal reagent for LC analysis should modify the phosphonic acid groups with a fluorescent label since such a reaction would be applicable to all bisphosphonates. Unfortunately, such a reaction has not yet been described for bisphosphonates. The bisphosphonate derivatives, suited for GC analysis, are all susceptible to deterioration in the presence of moisture and are consequently not suited for any (aqueous) LC separation; other derivatization reactions will be required. For example, for different alkylphosphonic acids, a fluorescent derivative, appropriate for LC analysis, has been synthesized using p-(9- anthroloxy)phenacyl bromide . Development of an analogous reaction with a bisphosphonate would be a very interesting option.
For the development of analytical separation methods for bisphosphonates several strategies have been followed, mainly based on LC, IEC and reversed-phase liquid chromatography (RPLC), often in combination with an ion-pairing agent, in particular. GC, and more recently capillary electrophoretic (CE) separations have also been described. The multiple ionic and adsorptive character of a bisphosphonate requires specific measures in the development of an analytical separation. All possibilities will be discussed in the next sections.
Since all bisphosphonates are strongly ionic at moderate pH, IEC is the first chromatographic technique taken into consideration. Chromatography of these anionic analytes should be possible on an anionexchange stationary phase if no irreversible adsorption occurs. To prevent adsorption, a stationary phase based on a polymeric resin is probably more suitable than one based on a silica matrix. Styrene   , methacrylate      , hydroxy ethyl methacrylate  and ethylvinylbenzene     , all polymers copolymerized with divinylbenzene, have been applied as stationary phase matrices for the IEC of bisphosphonates. The matrices are modified into an ion-exchange resin, often with quaternary ammonium functions, and are therefore suitable for the chromatography of relatively strong ionic species such as bisphosphonates. Drawbacks of IEC are the limited efficiency and a selectivity that is difficult to adjust. Probably because of the limited selectivity only three bioanalytical methods employed IEC. Clodronate has been determined in urine [9 43] and serum  and pamidronate  in urine and plasma by IEC. Usually, the adjustment of the selectivity in IEC is only possible for moderately basic or acidic ions by changing the pH. In other situations, such a change is only possible if other interactions besides the interaction with the ionic groups of the stationary phase occur. For example, organic ions can show hydrophobic interactions with a polymer based solid phase. However, recently, a new method, improving the adjustability of the selectivity in the IEC analysis of bisphosphonates, was introduced . By adding copper(II) ions, complexing with bisphosphonates, to
the eluent, the copper(II) concentration became an extra tool in selectivity control for this chromatographic technique. For pharmaceutical quality control purposes, where sensitivity and selectivity are less important compared to analysis in biological matrices, IEC is the most frequently published technique in bisphosphonate analysis     (Figure 1)   (Table II). The eluent is mostly an aqueous solution with one ion, often nitrate, competing with the anionic analyte, and a counter ion. Besides nitrate, also chloride , acetate  and trimesate  have been reported as the competing eluting ion.
Since the selectivity of RPLC is greater than that of IEC, this separation technique will probably be the first choice for bioanalytical bisphosphonate analysis if it is possible to give the bisphosphonate sufficient retention on a hydrophobic stationary phase. This can be difficult because of the ionic character of bisphosphonates; the application of an ion-pairing agent can therefore be very helpful. Also the adsorptivity of bisphosphonates can disturb an efficient separation; this can be prevented by specific additions to the eluent. Adsorption suppression agents that have been applied are trioctylamine  and different tetraalkylammonium   (Figure 2)         ion-pairing agents, EDTA  , citrate   (Figure 3)   and even etidronate     . Citrate was only applied in combination with polymer based [poly(styrene-divinylbenzene)] stationary phases, while an ion-pairing agent appeared to be suitable to both polymer and silica based stationary phases.
In order to elute an ionic, non volatile analyte, in the gas phase, derivatization prior to the separation is a prerequisite. By a modification of the polar sites of the molecule, containing active hydrogen atoms, into a more hydrophobic group, a more volatile analyte can be obtained (see section on derivatization). If this is accomplished, GC can be a very selective and sensitive separation method. However, for bisphosphonates this may be a problem since all known GC derivatization reactions are performed in a non-aqueous solvent. The limited number of reports on GC analysis of these analytes may be a result of difficulties in transferring the ionic bisphosphonate into such an organic solvent. From the four known GC methods for bisphosphonates, one bioanalytical assay was for clodronate in urine  (Figure 4) and one for pamidronate in serum and urine . The other reports comprised the pharmaceutical analysis of etidronate  and pamidronate  in water. All authors applied a poly(methylsilicone) stationary phase.
CE is a more recently developed analytical technique compared to chromatography. CE can offer a superior separation efficiency, due to the laminar flow profile, and is especially appropriate for ionic components. Currently, a few CE methods on bisphosphonates have been described; however, none of them was employed for biological samples. Zeller et al.  applied isotachophoresis for the analysis of pamidronate tablets in a poly(tetrafluoroethylene) capillary (0.5 mm) with chloride as the leading and acetate as the terminating ion (Figure 5). Pharmaceutical analysis of alendronate was performed with zone electrophoresis in a fused uncoated silica capillary (75 µm) with a nitric acid/copper(II) sulphate electrolyte . Shamsi and Danielson  electromigrated different polyphosphates and polyphosphonates (etidronate was one of them) with different ribonucleotide electrolytes in a 50 µm fused silica capillary, also by zone electrophoresis.
The purpose of a detector is to register the amount or concentration of an analyte leaving the chromatographic column by measuring a physical property of the eluate. This can be done optimally when the detector is specific, sensitive and linear. Popular LC, CE and GC detectors are usually linear over at least three orders of magnitude; both the specificity and sensitivity can vary considerably between detectors. For GC analysis the thermoionic detector, also called nitrogen-phosphorous detector, is specific for phosphorus and sensitive as well . Mass spectrometric (MS) detection with selected ion recording will also be very specific and sensitive and was applied for the bioanalysis of clodronate  (Figure 4). Also relatively specific but less sensitive is the flame photometric detector (FPD), applied to pamidronate derivatives  . The very common flame ionisation detector (FID) is very unspecific and comparable in sensitivity to the FPD . The FID was applied for the pharmaceutical analysis of etidronate , where both sensitivity and specificity are less critical compared to bioanalysis. If an adequate derivatization reagent, containing halogen atoms, is chosen, then application of the most sensitive GC detector, the electron capture detector, is an interesting option. However, all byproducts of the derivatization reaction will also be detected in this procedure. This would not happen with a detector, showing selectivity for a native property of the analyte, for example single ion-MS or FPD. In LC and CE a direct sensitive detection is only possible for a few bisphosphonates. These bisphosphonates have specific detectable properties like the UV-absorbing phenyl ring in tiludronate [((4-chlorothiophenyl) methylene)bisphosphonate] , the fluorescent imidazopyridyl group in YM-529  and the oxidizable cycloheptylamino group in incadronate  Figure 2. For other bisphosphonates derivatization is required to obtain low detection levels. A fluorescent derivative   (Figure 3)           is often preferred, as this type of derivative is in general the most sensitivily detectable. A derivative detectable with electrochemical detection can be an alternative . When low detection limits are not an important aim, for example in pharmaceutical assays, non specific detection methods can be applied to non derivatized bisphosphonates: conductivity    (Figure 5)], refractive index (RI)     or indirect UV absorbance   (Figure 1) .
AUTOMATIC SAMPLE HANDLING
As described previously, sample treatment is inextricably bound up with the bioanalysis of bisphosphonates. If, after method development, this treatment becomes too laborious, automation may be considered to save time, enlarging the sample throughput and, possibly, improving the reproducibility of the assay. Instruments for automated sample handling can generally be divided into three types. In order of decreasing investment and analytical possibilities, these instrument types are: robotic systems, robotic workstations and extended-capability autosamplers  . Robotic systems are characterized by their open architecture and infinite possibilities. Typically, robotic workstations are designed for SPE; however, some of these instruments can also be applied to other advanced tasks such as filtration, liquid- liquid-extraction and weighing . Extendedcapability autosamplers are generally only suited to relatively simple tasks such as injection, dilution and heating, although one or two single more advanced tasks may also be possible . Kline et al.   were the first to describe precolumn automation in bisphosphonate analysis, applying it to alendronate analysis in urine and plasma. An autosampler was employed for addition of buffer and reagent, in order to facilitate the derivatization reaction in the sample vial. This method was also applied in an assay for pamidronate in bone . Recently, Sparidans et al.   succeeded in automating also SPE and liquid-liquid-extraction, in addition to derivatization, in their bioassays for pamidronate and olpadronate. Precipitation, however, is extremely difficult to automate. When the liquid phase has to be processed further after precipitation, as in a procedure for protein precipitation, filtration can play a role in separation of the liquid phase from the precipitate. Unfortunately, further analysis of the precipitate (as in a procedure for co-precipitation of a bisphosphonate with calcium phosphate, the calcium precipitation method described)