The History Of The Method Development

Published Date: 02 Nov 2017

The first step in method development is to do extensive literature survey of the molecule studied to understand the physicochemical properties of the molecule. Chromatographic conditions can be developed in isocratic or gradient mode to achieve good chromatographic retention, resolution and signal to noise ratio (Table 1.1). Depending on the number of active components to be resolved or separated, the mode of run can be determined. If the number of components is large or the pKa values of components are wide apart then gradient mode is preferred over isocratic mode. In deciding whether a gradient would be required or whether isocratic mode would be adequate, an initial gradient run is performed and the ratio between the total gradient time and the difference in gradient time between the first and last component are calculated. When the calculated ratio is <0.25, isocratic is adequate; when the ratio is >0.25, gradient would be beneficial for the separation of complex mixture and when there are many compounds or degradation products, a long gradient run may be needed.

Table 1.1: General Experimental Conditions for an Initial HPLC Run

Initial parameters

Chromatographic variables

Neutral compounds

Ionic-acidic compounds

(carboxylic acids)

Ionic-basic compounds

(amines)

Column Dimension

(length, ID)

Stationary phase

Particle size

5cm x 0.46cm

C18 or C8

5μm or 2.5μm

5cm x 0.46cm

C18 or C8

5μm or 2.5μm

5cm x 0.46cm

C18 or C8

5μm or 2.5μm

Mobile phase

Solvents A and B

%B (organic)

isocratic

%B (organic)

gradient

Buffer-acetonitrile

75%

5%/95%

Buffer-acetonitrile

75%

5%/95%

Buffer-acetonitrile

75%

5%/95%

Buffer Type

Concentration

Ammonium sailts/formic acid

10 mM/0.1%

Ammonium sailts/formic acid

10 mM/0.1%

Ammonium sailts/formic acid

10 mM/0.1%

Flow rate

0.8-1.5 mL/min

0.8-1.5 mL/min

0.8-1.5 mL/min

Temperature

Ambient to 40°C

Ambient to 40°C

Ambient to 40°C

Sample size

Volume

10μL-25μL

10μL-25μL

10μL-25μL

In general, one begins with reversed phase chromatography, when the compounds are hydrophilic in nature with many polar groups and are water soluble. The organic phase concentration required for the mobile phase can be estimated by gradient elution method. For aqueous sample mixtures, the best way to start is with gradient reversed phase chromatography. Gradient can be started with 5-10 % organic phase in the mobile phase and the organic phase concentration can be increased up to 100% within 2-3 min. Separation can then be optimized by changing the initial mobile phase composition and the slope of gradient according to the chromatogram obtained from preliminary run. The initial mobile phase composition can be estimated on the basis of where the compounds of interest is eluted, namely, at what mobile phase composition, retention time and the pKa of the component. Changing the polarity of the mobile phase will alter elution of drug molecules. The elution strength of a mobile phase depends upon its polarity, the stronger the polarity, higher is the elution. Ionic samples (acidic or basic) can be separated, if they are present in undissociated form. Dissociation of ionic samples may be suppressed by proper selection of pH. The buffer selected for a particular separation should be used to control pH over the range of pKa 1.5. pH of the buffer should be adjusted before adding organic (Snyder et al, 1997).

Method optimization can be initiated after a chromatogram with qualifying parameters has been obtained. A qualifying chromatogram is the one which has, symmetrical peaks, a good separation, less peak width and a reasonable run time. The peak resolution can be increased by using a more efficient column (column with higher theoretical plate, N), which can be achieved by using a column of smaller particle size, or a longer column. These factors, however, will increase the analysis time. Flow rate does not influence resolution, but it has a strong effect on the analysis time.

Selection of Mobile phase

Buffered mobile phases can be used to develop rugged methods, if the analysed sample consists of ionic or ionizable compounds. In nonbuffering mobile phases, based on the nature of test compound, a pH change in ±0.1 pH units can have a significant effect on the method performance. On the other hand properly used buffer allows controlling the pH easily. Buffer works best at the pKa of its acid. At this pH, the concentration of the acidic form and the basic form of the buffering species is equal, and the buffering capacity is maximum. For methods developed on LC-MS/MS, ammonium salt buffers can be used as mobile phase, because of their volatile nature. Ammonium salt buffers can be used in the concentration range of 2-10mM, but in few cases a maximum of 50mM concentration can be used. Few compounds tend to form ammonium adducts in the ionisation source in positive mode of analysis. Basic compound will usually show enhanced signal by lowering pH of mobile phase in LC/MS/MS.

In many cases, silanophillic interactions cause tailing, mostly due to ion exchange interaction. This can usually be reduced or suppressed by the use of amine-based buffers or by using acidic mobile phases, or a combination thereof. Whenever buffers or other mobile phase activities are used, check the solubility in mobile phase. This is especially true for gradient applications. Of all the organic modifiers available, acetonitrile is the first choice of organic modifier for analysis in reverse-phase separations. Acetonitrile based mobile phases can give an up 2-fold lower pressure drop than methanol based mobile phase at equal flow rate. This means that column efficiency is higher. The elution strength increases in the order methanol, acetonitrile and tetrahydrofuran. The retention changes by roughly 10 % for every 1 % change in the concentration of organic modifier.

Role of pH

pH of mobile phase affects the resolution and sensitivity in LC-MS/MS methods. Improper pH of the mobile phase leads to assymetrical peak shapes and decreased sensitivity. Asymmetrical peaks creates difficulties in quantitative analysis for achieveing lower quantification limits, lower Relative Standard Deviation (RSD) between injections and reproducible retention times. So pH of the mobile phase is very critical in the process of method development.

Role of Buffer

In general for reverse phase applications, pH of the mobile phase will be used in between 2.0-7.0. Buffer pH that was correctly chosen will keep the ionizable functional groups in a single state, that can be either ionic or neutral. Some compounds will impart pH to the sample solution injected that can damage the column if the extreme pH conditions exists, in such cases the buffer will fastly neutralise the pH of the injected solution to desired pH. In LC-MS/MS methods buufer pH, buffer concentration will impact the sensitivity of the method, so a careful selection of the buffer should be investigated.

Selection of Column

Column selection is a very critical component in the process of method development, as few critical sepaarations, symmetric peaks, recovery and retention depends on the column chemistry selected for the compounds under method development. The main goals of column selection should be aimed at selectivity, sensitivity, signal to noise ratio and reproducibility. Commonly used reversed phase columns are C18 (octadecylsilane,), C8 (octylsilane,) phenyl and cyano. Columns with different chemistry (C18, C8, phenyl and cyano), different lengths (50, 75, 100 mm), different internal diameter (4.6, 3.0, 2.0 mm), different particle size (5, 3.5, 2 µm) should be tested in method development to appropriately pick the best combination. .

Role of temperature

Variations in room tempearture will have significant effect on HPLC separations and retention. In general retention time of neutral compounds decrease with an increase in temperature, but ionised compounds retention will not have significant impact from the changes in temperature. An increase in temperature by 1°C will decrease the retention time of compounds by 1-2%. Due to these consequences, it is always recommended to place the column in thermostated conditions during method development, validation and routine screening.

Role of Flow rate

Changes in flow rate, can be more useful for isocratic separations than gradient separation to increase the separation. Using lesser flow rates will result in low column back pressure, but the disadvantage with using low flow rates is increase in run time. Method optimization can be initiated only after a justifiable chromatogram has been obtained.

Selection of Internal Standard

Internal satndard is a compound added externally to a sample in known concentration to facilitate quantitative determination of the sample components, as it accounts for processing and instrumental variations. Isotopically labeled compounds can best serve as internal standard, due to their similarity in physicochemical properties to the parent compound. Isotopically labeled internal standard will have similar chromatographic retention, recovery, response in LC-MS/MS.

Sample preparation techniques

Protein precipitation (PPT)

It is a rapid, nonspecific method that can be utilized for sample clean-up in a high-throughput, automated manner. PPT-based purification relies on reduced solubility of proteins and highly polar matrix components in aqueous-organic solvent solutions. Acetonitrile, methanol or acetonitrile-methanol mixtures are most commonly used for PPT. More than 90% of plasma proteins can be removed from samples using PPT when the plasma to organic solvent ratio is at least 1 to 2.5 (Chambers et al., 2007; Polson et al., 2003).

Liquid-liquid extraction (LLE)

LLE is based on the partitioning of an analyte into two separate liquids. The technique works by taking advantage of the differential solubility of an analyte in two immiscible liquids. One of the phases usually is water or a buffer solution or biological matrix, while the other is an organic solvent. Selection of the proper organic solvent to obtain maximum recovery should be based on the analyte’s solubility in the particular solvent (Chambers et al., 2007). LLE alone as an extraction technique might not be suitable enough for getting rid of the diversified matrix effects as some of the matrix components are even soluble in the organic solvents used for extraction.

Solid phase extraction (SPE)

SPE methods rely on the affinity of an analyte for a stationary phase and are often used to isolate analyte(s) of interest from a wide range of matrices including urine, blood, tissue homogenates, etc. Depending on the properties of the analyte and the solid phase, either the analyte of interest is retained while the unwanted matrix components elute with the solvent wash. Or the unwanted matrix components are retained and the analyte elutes with the solvent wash. There are numerous SPE stationary phases available, including normal phase, reversed phase, and ion exchange (Chambers et al., 2007; Supelco, 1998). In some cases, where the physicochemical properties of the test article and formulation vehicle are similar, SPE might not be a good extraction technique as the final extracts will have both test articles of interest and matrix effect causing components. Therefore, SPE is less useful for a high-throughput analysis of a diverse set of compounds encountered in the early stages of the drug discovery but is widely used for clinical sample analysis (Terence G. Hall e al).

Method Validation

Once an LC-MS/MS method (including the proper sample extraction method) has been developed, it is necessary to evaluate the performance of the method, especially when the developed method will be utilized for quantitative purposes. Although there are many different standards for method validation, the most commonly accepted one in the pharmaceutical industry in United States is the "Guidance for Industry Bioanalytical Method Validation" proposed by FDA. According to FDA, several factors about the method and the analyte have to be considered when validating a bioanalytical method. These factors include selectivity, recovery, calibration curve, accuracy, precision, and stability. Besides, as the matrix of the biological samples, even the matrix of the post-extract samples, may affect the signal of the analyte, evaluation of matrix effect has also become a must in the method validation.

Selectivity

This measure studies how specific the bioanalytical method is toward the analyte. The interferences coming from same type of blank biological samples, yet from at least six different sources have to be tested. The signal of the interference caused by nonspecific responses should be evaluated at the level of lower limit of quantification(LLOQ). In another word, the non-specific response of the detector should not affect the accurate quantification of the LLOQ.

Calibration curve

The calibrators must be prepared in the same biological matrix as the real samples. Usually, the calibrators should be prepared by spiking know amount of analyte into the biological matrix. For a linear calibration curve, at least six non-zero calibrators should be included in the curve. A double-blank sample with no addition of analyte or the internal standard (IS) should be included. Besides, a zero sample, with no addition of analyte, yet containing same concentration of IS as all the non-zero calibrators, should also be included. The accuracy and precision of each calibrator should be evaluated. The accuracy of each calibrator is determined by the percent error of the calculated concentration. Here (Analyte)mean: represents the measured concentration of the analyte from the calibration curve. (Analyte)nomi: indicates the nominal concentration of the analyte. The inter-assay precision of each calibrator is determined by the coefficient of variation (CV %) from several measurements toward the same calibrator.

(Equation 1.1)

Sometimes, the same calibration curve is repeated for several times on different days, so that the inter-assay precision of the calibrators can be determined. By dividing the standard deviation of the calculated values with the average of the calculated values, the inter-assay precision can be obtained. For any point on the calibration curve, except the LLOQ, the accuracy and precision should be within ±15%. For LLOQ, the values should not exceed ±20%.

(Equation 1.2)

Accuracy and Precision

The calculations of the accuracy and precision are the same with those described above. However, to evaluate the accuracy and precision of a method, a set of quality control (QC) samples are needed. These QC samples usually include at least three concentration levels (low, medium, and high). For each concentration level, five parallel replicates should be prepared. Since the accuracy and precision at the LLOQ should also be evaluated, if the low concentration QC samples (LQCs) are not the same with the LLOQ, another five replicates of LLOQ should be prepared. During real sample analysis, there is possibility that some of the real samples possess concentrations either higher or lower than the upper or lower limit of quantification. In these occasions, diluted or concentrated quality control samples are also need for the accuracy and precision studies. The acceptable criteria of accuracy and precision for the QC samples are, again, within ±15%, except at LLOQ as ±20%.

Recovery

It is an evaluation of the effectiveness of the extraction methods. It can be obtained by comparing the signals of the analyte in the extracted sample and in the pure authentic analyte solution spiked in the post-extraction blank matrix. To determine the recovery of the extraction method accurately, at least three concentration levels (i.e., a low, a medium, and a high) throughout the calibration range should be evaluated. At each concentration level, three replicates of the samples should be prepared. The mean value and the standard deviation of the measurements should be recorded and reported.

Matrix effect

It is an evaluation of the effectiveness of both the extraction methods and LC methods, because if the extraction method or LC failed to provide enough separation between the analyte and the interferences, the signal of the analyte may be significantly suppressed or, sometimes, enhanced. Matrix effect is usually calculated by comparing the signals of the analyte in the pure authentic solution spiked in the postextraction blank matrix with that in the pure authentic solution along. Like the determination of recovery, at least three concentration levels are needed, and three replicates are required for each concentration level. The mean and standard deviation of the measurements should be recorded and reported.

Stability

The stability studies provide important information on sample handling and storage. The most common stability studies include the following categories: short-term stability, long-term stability, freeze and thaw stability, post-preparative stability, and stock solution stability. The short-term, long-term, and freeze and thaw stabilities illustrate the stability of the analyte in the biological matrix. The post-preparative stability analyzes the stability of the analyte in the post-extraction sample matrix. The stock solution stability measures the stability of the analyte in high concentration pure stock solutions. For each stability study, at least two concentration levels, a low and a high, are needed. At each concentration level, three parallel replicates should be prepared. The signals of the analyte in the samples after incubation will be compared with those freshly made samples. The degradation of the analyte can then be clearly indicated by the signal loss.

Short-term stability

These studies are usually carried out under room temperature. The biological matrix spiked with known concentrations of analyte will be left on bench top or in a place that away from direct light exposure. The samples are usually kept under this condition for 4 to 24 h. However, the study time can be adjusted accordingly based on the actual need in the real sample analysis.

Long-term stability

In this type of studies, the spiked samples should be kept in a desired storage place, such as a refrigerator (4oc) or in a freezer (-20 or -80 oc). The total study time should be longer than the time span between the collection time of the first sample and the analysis time of the last sample. The most common time span is from 1 to 6 months.

Freeze and thaw stability

At least three freeze and thaw cycles should be carried out for this type of studies. For each cycle, the samples should be frozen in the desired storage temperature (e.g., -20 or -80 oc) for 12-24 h. Then, the samples should be thawed under room temperature without assistance.

Post-preparative stability

After the samples are processed, they usually will be kept in an auto sampler, waiting for analysis. Therefore, the stability of the analyte and the internal standard in the processed samples should also be evaluated. The study time should not be shorter than the total running time of a whole batch of sample.

Stock solution stability

For the stock solutions, at least two kinds of stabilities must be studied: short-term stability at room temperature, as well as freeze and thaw stability. At least 6 hours storage at room temperature should be included in the short-term stability studies; at least three freeze-and-thaw cycles should be carried out in the freeze and thaw stability studies.

Matrix Effect

Matrix effects can cause significant errors in precision and accuracy, thereby invalidating the assessment of pharmacokinetic results based on the LC–MS/MS assays. When matrix effects cause differential suppression or enhancement between calibration samples and study samples, the accuracy of the assay results will be significantly affected (Walter A. Korfmacher). Matrix effect affects the reproducibility, sensitivity and reliability of the analytical techniques.

Formulation excipients used in early drug discovery for solution or suspension formulations are known to interfere with analyses. These excipients are present at high concentration (>1mg/mL) in earlier pk sampling time points. Some formulation vehicles cause 80-90% ion suppression when administered in both intravenous and oral administration routes (XU et al., 2005; Larger et al., 2005; Shou WX et al., 2003; Tong et al., 2002).

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is a highly specific and sensitive analytical technique that has become the industry standard for quantifying drugs, metabolites, and endogenous compounds in biological matrices. The technique is widely used because of its ability to accurately quantitate analytes of interest with minimal sample clean-up and rapid LC separation. Despite these advantages, LC-MS/MS methodology occasionally encounters problems, some of which are caused by matrix effects. The "matrix" refers to all components in the sample other than analyte(s) of interest (Terence G et al). Matrix effects are defined as "interference from matrix components that are unrelated to the analyte".

Matrix effects can arise from a number of matrix components including, but not limited to:

Endogenous biological components such as phospholipids, carbohydrates, and endogenous metabolites.

Residual formulation components used in the preparation of formulations

An interaction between the analyte of interest and the matrix, such as covalent binding to plasma proteins or the enzymatic degradation of a prodrug

Co-eluting drug metabolites

Concomitant medications

Mobile phase additives

Matrix effect affects the reproducibility, sensitivity and reliability of the analytical techniques.

Apart from the much spoken endogenous components causing matrix effects, exogenous components (formulation excipients) used in the preparation of formulations were also concern as they could potentially cause suppression or enhancement of the analyte and internal standard which in turn will have impact on the accuracy of measured concentrations. A lot variety of formulation excipients ranging from cosolvents, complexing agents, lipid based vehicles and surfactants will be used in the preparation of formulations at preclinical level.

The exact mechanisms by which matrix components cause ionization suppression or enhancement are not known. Matrix effects arise at the interface between the LC system and the MS system (Richard King et al., 2000)

Various mechanisms by which the matrix components cause matrix effects are as follows:

Charge competition between analyte and matrix components (Bennett and Liang, 2004; Chambers et al., 2007)

Change in droplet surface tension leading to formation of large droplets and insufficient desolvation (Bonfiglio et al., 1999; King et al., 2000)

Preferential ion evaporation due to matrix components gathering at droplet surface

Change in mass of analyte ion due to ion pairing and adduct formation

Co precipitation with nonvolatile matrix components (Van Hout MW et al., 2003)

Gas phase deprotonation

Electrospray Ionisation (ESI) is more prone to matrix effects than the other sources. In the ESI source, analytes must acquire a charge in solution and then successfully transition to gas phase while maintaining their charge. The acquisition of charge in the solution phase and successful transitioning to the gas phase makes the ESI source the most vulnerable to matrix effects when compared to either APCI or APPI (Jessome Lori Lee et al, 2006; King et al., 2000; Trufelli et al., 2011).