Calibration of analyte amount in mass spectrometry

Abstract

A method of determining the concentration or quantity of an analyte of interest comprises analysing a set of analytical samples by mass spectrometry, ion mobility separation or optical spectroscopy. O

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority from and the benefit of United Kingdom patent application No. 2408094.7 filed on 7 June 2024. The entire contents of this application are incorporated herein by reference. FIELD OF THE INVENTION The present invention relates generally to mass spectrometry, and in particular to techniques for quantifying the amount of an analyte that is present in a sample. BACKGROUND It is known to mass analyse samples using mass spectrometry in order to determine the quantity or concentration of an analyte in the sample. In order to determine the quantity or concentration of the analyte in the sample it is necessary to use a calibration relationship that relates the ion signal for the analyte that is detected by the mass spectrometer to the quantity or concentration of the analyte in the sample. In order to obtain the calibration relationship it is typical to mass analyse various calibration samples. Although this approach is effective, the calibration samples take additional time to mass analyse. Also, when samples are being analysed in batches of a restricted size, the calibration samples take up space in the batch that cannot then be used to analyse analytical samples. This is particularly problematic for batches that are restricted to having relatively few samples therein. It is known to provide a set of different stable isotope labelled calibrator (SILC) compounds in each analytical sample, rather than providing them as separate calibration samples. However, this approach is costly due to the high cost of manufacture of SILCs. SUMMARY From a first aspect the present invention provides a method of determining the concentration or quantity of an analyte of interest in an analytical sample comprising: mass analysing a first set of analytical samples, wherein one of the analytical samples contains a plurality of calibrators at different known concentration levels and another of the analytical samples does not contain at least one of said calibrators, wherein said mass analysing obtains an ion signal for an analyte of interest in each of the analytical samples, and wherein said mass analysing obtains an ion signal for each known concentration level of said calibrators in said one of the analytical samples; determining a calibration relationship based on said plurality of different known concentration levels of the calibrators and their respective ion signals obtained from mass analysing said one of the analytical samples; and determining the concentration or quantity of the analyte of interest in at least one of said set of analytical samples using the ion signal detected for the analyte of interest in that analytical sample and the calibration relationship. As the different known concentration levels of the calibrators are provided in the same analytical sample, rather than in separate samples as is conventional, the present invention is able to mass analyse the analyte of interest and also obtain the mass spectral data needed for determining the calibration relationship in an efficient manner. Also, as all of the plurality of calibrators are only provided in one of the analytical samples, only a relatively small amount of calibrator material is needed to perform the method. Although it would be possible to include all of the different known concentrations levels in each of the analytical samples, this requires a relatively large amount of calibrator material. It would also require each concentration level to be analysed in each sample, thus requiring a relatively large amount of analysis per sample. The inventors of the present invention have recognised that the full set of calibrators may be provided in only a subset of the analytical samples in order to obtain the calibration relationship, whilst still providing an effective calibration relationship that can be used for the other analytical samples that do not contain all of the calibrators. The method may determine the calibration relationship based on all of said plurality of different known concentration levels of the calibrators and their respective ion signals obtained from mass analysing said set of analytical samples. Each analytical sample may contain an unknown quantity or concentration of the analyte of interest. Said set of analytical samples may be formed from consecutively mass analysed analytical samples. The analytical samples may be mass analysed by the same mass spectrometer. The set of analytical samples may have an unknown quantity or concentration of the analyte of interest in them and may be mass analysed consecutively without mass analysing any other samples in between them. Alternatively, one or more other type of sample, such as a quality control sample, may be mass analysed between the consecutively mass analysed analytical samples. Said one of the analytical samples that contains the plurality of calibrators may be either: i) the first analytical sample to be mass analysed in said set of analytical samples; ii) the final analytical sample to be mass analysed in said set of analytical samples; or iii) an analytical sample that is mass analysed substantially midway between the first and final analytical samples to be mass analysed in said set of analytical samples. Only a single analytical sample in said set of analytical samples may contain all of said plurality of calibrators. Said set of analytical samples may contain >3, >4, >5, >6, >8, >10, >20, >30, >40, >50, >60, >70, >80, >90, or >100 analytical samples. However, it may be preferred that the set of analytical samples contains relatively few analytical samples, such that the calibration relationship remains valid if it is applied to all of those analytical samples in order to determine the concentration or quantity of analyte of interest therein. Accordingly, the set of analytical samples may be <70, <60, <50, <40, <30, <20, <10, or <5 analytical samples. The calibration relationship may be used to determine the concentration or quantity of the analyte of interest in each of the analytical samples in said set of analytical samples. Where only a single analytical sample in said set of analytical samples contains all of said plurality of calibrators, the remaining samples in said set of analytical samples may have none of said calibrators therein. Alternatively, the remaining samples in said set of analytical samples may have only one of said calibrators therein, which is at the same concentration in all of said remaining samples. The calibrator in the remaining samples may be used as an internal standard. For example, when each of the remaining samples is mass analysed, a scaling factor may be obtained based on the ion signal (e.g. peak area or height) for the calibrator in that sample and the ion signal (e.g. peak area or height) for the analyte of interest in that sample, and the ion signal (e.g. peak area or height) for the analyte of interest may then be multiplied by the scaling factor. The method may comprise estimating a range of concentrations that the analyte of interest is expected to have in the analytical samples, wherein said different known concentration levels of the calibrators include a first concentration level above said range and / or a second concentration level below said range. The method may comprise estimating a range of concentrations that the analyte of interest is expected to have in the analytical samples, wherein said different known concentration levels of the calibrators includes at least one concentration level within said range. Each of the calibrators may be a compound that produces an ion signal when mass analysed that varies according to its concentration in substantially the same way that the ion signal for the analyte of interest varies according to its concentration. Each of said calibrators may be a compound that is the same as the analyte of interest, except that one of more of the atoms in its chemical structure has been substituted for a different, stable isotope of that atom. Such calibrator compounds are referred to herein as stable isotope labelled calibrator (SILC) compounds. However, other compounds may be used as the calibrators instead, such as a compound that is different to the analyte of interest but is in the same family of compounds as the analyte of interest. The plurality of calibrators may have different molecular masses. The mass spectrometer may be operated to always mass analyse the calibrators if they are present, or it may be operated to not mass analyse the calibrators in some of the analytical samples. For example, the mass spectrometer may be programmed not to mass analyse the calibrators for samples that the calibrators have not been added to. The method may further comprise performing a cycle that comprises: a) mass analysing a further set of analytical samples, wherein one of these analytical samples contains said plurality of calibrators at different known concentration levels and another of these analytical samples does not contain at least one of said calibrators, wherein this mass analysing obtains an ion signal for the analyte of interest in each of the analytical samples in the further set, and wherein the mass analysing obtains an ion signal for each known concentration level of said calibrators in said further set of the analytical samples; b) updating said calibration relationship, or determining a new calibration relationship, using the ion signal obtained for each known concentration level of said calibrators in step a); and c) determining the concentration or quantity of the analyte of interest in at least one analytical sample using the ion signal detected for the analyte of interest in that analytical sample and the updated or new calibration relationship. By updating said calibration relationship it is meant that the calibration relationship obtained prior to said cycle being performed is modified using the ion signals obtained for the calibrants during said cycle. In other words, the updated calibration relationship is formed using the ion signals obtained for the calibrants prior to said cycle being performed and also the ion signals obtained for the calibrants during said cycle. In contrast, by said new calibration relationship it is meant that the calibration relationship is formed using only the ion signals obtained for the calibrants in said cycle. The further set of analytical samples may have any of the features described in relation to the first set of analytical samples. Step c) may comprise determining the concentration or quantity of the analyte of interest in at least one analytical sample in said further set of analytical samples using the ion signal detected for the analyte of interest in that analytical sample and the updated or new calibration relationship determined in step b). For example, step c) may comprise determining the concentration or quantity of the analyte of interest in all of the analytical samples in said further set of analytical samples using the ion signal detected for the analyte of interest in each of those analytical samples and the updated or new calibration relationship determined in step b). The updated or new calibration relationship may be used only to determine the concentration or quantity of the analyte of interest in analytical samples that were mass analysed in step (a). The method may comprise repeating said cycle a plurality of times. Each time the cycle is performed, the set of analytical samples that is mass analysed in that cycle may have any of the features described in relation to the first set of analytical samples. In the embodiments that update the calibration relationship, each time the cycle is performed the calibration relationship from step b) of the preceding cycle may be updated using only a preselected number of most recently obtained ion signals for each of the different concentration levels. The method may therefore discard older calibration data that is less likely to be accurate for the analytical samples that have been analysed more recently. Each time the cycle is performed, the updated or new calibration relationship may be used to determine the concentration or quantity of the analyte of interest in analytical samples that were mass analysed in step (a) of that cycle. For example, the updated or new calibration relationship may be used only to determine the concentration or quantity of the analyte of interest in analytical samples that were mass analysed in step (a) of that cycle. Each time the cycle is performed, step c) may comprise determining the concentration or quantity of the analyte of interest in at least one analytical sample in the previously mass analysed set of analytical samples using the ion signal detected for the analyte of interest in that analytical sample and the updated or new calibration relationship determined in step b). The cycle may be repeated for a pre-selected number of cycles during each of which step b) updates the calibration relationship obtained in the previous cycle, and then the cycle is performed a further time in which a new calibration relationship is determined that does not use the ion signals for the calibrants that were obtained in any preceding cycles. The cycle may only be repeated for up to a pre-selected number of cycles or for a pre-selected duration of time. Where said cycle is performed one or more times, all of the sets of analytical samples may have the same number of analytical samples therein. Alternatively, different sets of analytical samples may have different numbers of analytical samples therein. The number of analytical samples in each set of analytical samples may be the same as the number of calibrators having different known concentration levels. The method may comprise providing a batch of samples containing analytical samples, wherein either: i) the only analytical samples in the batch are said first plurality of analytical samples; or ii) the method comprises performing or repeating said cycle until all of the sets of analytical samples in the batch have been mass analysed. In all embodiments that provide a batch of samples, the batch may have a predetermined maximum number of samples therein. For example, the batch may have a number of samples that is <100, <90, <80, <70, <60, <50, <40, <30, <20, or <10. The batch of samples may be provided in a sample plate having a predetermined number of sample wells therein. The batch of samples may only include said different known concentration levels of the calibrators in the first analytical sample that is mass analysed, in the final analytical sample that is mass analysed, and in a further analytical sample that is mass analysed between the first and final analytical samples; optionally wherein the further analytical sample is mass analysed substantially midway between mass analysing the first and final analytical samples. The method may comprise providing a plurality of batches of samples that include analytical samples and performing the method described above on each of the batches, wherein either: (i) the different known concentration levels of the calibrators that are mass analysed first in any given batch are used to form a new calibration relationship that is used to determine the concentration or quantity of the analyte of interest in one or more analytical sample in that batch; or (ii) the different known concentration levels of the calibrants that are mass analysed first in any given batch are used to update the calibration relationship obtained at the end of analysing the previous batch, and the updated calibration relationship is used to determine the concentration or quantity of the analyte of interest in one or more analytical sample in said given batch. However, it is contemplated that the samples may not be mass analysed in batches and may instead be mass analysed as a stream of samples arriving at the mass analyser. The analytical samples may be mass analysed using liquid chromatography mass spectrometry. Optionally, each of the analytical samples is mass analysed using tandem mass spectrometry by monitoring an MRM transition for said analyte of interest, and at least the analytical samples containing the calibrators are mass analysed using tandem mass spectrometry by monitoring an MRM transition for each of the calibrators. The analytical sample may be a biological sample, such as from a patient. For example, the sample me be, or comprise, blood, blood plasma, blood serum, urine, stool, saliva or cerebrospinal fluid. The analyte of interest may be a steroid hormone, a vitamin, a drug of abuse (e.g. an opiate), a therapeutic drug such as an immunosuppressant, an anti-epileptic drug, an anti-infective drug, a steroid, an anti-fungal drug, or an amino acids etc. Although the method has been described as determining the concentration or quantity of a single analyte of interest, the method described herein may be used to determine the concentration or quantity of multiple different analytes of interest in each analytical sample. The number of calibrators having different known concentration levels may be between four and seven calibrators. However, it is contemplated that fewer or a greater number of calibrators having different known concentration levels may be used. For example, at least three, or at least seven, calibrators having different known concentration levels may be used. The method may be performed by automated machinery that: (i) receives samples; (ii) adds said plurality of different known concentration levels of the calibrators to one of the analytical samples so as to form said set of analytical samples; and (iii) transfers the set of analytical samples to one or more mass spectrometer so as to then perform the method described above. Each of the analytical samples that is received at the automated machinery may have an identifier thereon that represents the type of sample that it is and / or a test that is required to be performed on that sample; and the automated machinery may read the identifier and select analytical samples of the same type and / or that are to undergo the same test and performs step (ii) on these analytical samples. The automated machinery may group the selected analytical samples into one or more batches of analytical samples, each of which may contain one or more of said sets of analytical samples. The identifier may be a barcode and the automated machinery may be a barcode reader. However, it will be appreciated that may other types of identifier and automated reader may be used. Although embodiments have been described in which the plurality of calibrators at different known concentration levels are added to one or more analytical sample, it is contemplated that alternatively, or additionally, the calibrators may be added to each of one or more quality control samples. Accordingly, from a second aspect the present invention provides a method of determining the concentration or quantity of an analyte of interest in an analytical sample comprising: mass analysing a first set of samples that comprises a quality control sample and a plurality of analytical samples, wherein said quality control sample contains a plurality of calibrators at different known concentration levels and at least some of the analytical samples do not contain at least one of said calibrators, wherein said mass analysing obtains an ion signal for an analyte of interest in each of the analytical samples, and wherein said mass analysing obtains an ion signal for each known concentration level of said calibrators in said quality control sample; determining a calibration relationship based on said plurality of different known concentration levels of the calibrators and their respective ion signals obtained from mass analysing said quality control sample; and determining the concentration or quantity of the analyte of interest in at least one of said analytical samples using the ion signal detected for the analyte of interest in that analytical sample and the calibration relationship. The quality control sample contains a known amount of the analyte of interest, e.g. a known concentration or quantity of the analyte of interest has been added to it prior to it being mass analysed. In contrast, the concentration or quantity of the analyte of interest in the analytical samples is unknown prior to the mass analysis. The first set of samples may include more than one quality control sample, and the known amount of the analyte of interest may be different in the different quality control samples. The analyte of interest described herein may be an analyte that is found in a human patient or animal, whereas the calibrators may not naturally be found in a human patient or animal (e.g. in a significant quantity). The method according to the second aspect of the invention may have any of the features described in relation to the first aspect of the invention, except that instead of all of the plurality of calibrators being provided in an analytical sample in each set of samples, these calibrators are provided in a quality control sample in each set of samples. Accordingly, the set of samples according to the second aspect of the invention may have the features described above in relation to the set of analytical samples in the first aspect of the invention. For example, the set of samples may be formed from consecutively mass analysed samples. The quality control sample that contains the plurality of calibrators is either: i) the first sample to be mass analysed in said set of samples; ii) the final sample to be mass analysed in said set of samples; or iii) a sample that is mass analysed substantially midway between the first and final samples to be mass analysed in said set of samples. Only a single sample in said set of samples may contain all of said plurality of calibrators. Said set of samples may contain >3, >4, >5, >6, >8, >10, >20, >30, >40, >50, >60, >70, >80, >90, or >100 samples. However, it may be preferred that the set of samples contains relatively few samples, such that the calibration relationship remains valid if it is applied to all of the analytical samples therein in order to determine the concentration or quantity of analyte of interest. Accordingly, the set of samples may be <70, <60, <50, <40, <30, <20, <10, or <5 samples. The calibration relationship may be used to determine the concentration or quantity of the analyte of interest in each of the analytical samples in said set of samples. All of the analytical samples in the set of samples may not contain said at least one of the calibrators. All of the analytical samples in the set of samples may not contain any of the calibrators. Alternatively, all of the analytical samples in the set of samples may contain one of the calibrators for use as an internal standard. For example, when each of the analytical samples is mass analysed, a scaling factor may be obtained based on the ion signal (e.g. peak area or height) for the calibrator in that sample and the ion signal (e.g. peak area or height) for the analyte of interest in that sample, and the ion signal (e.g. peak area or height) for the analyte of interest may then be multiplied by the scaling factor. The method may further comprise performing a cycle that comprises: a) mass analysing a further set of samples that comprises a quality control sample and a plurality of analytical samples, wherein this quality control sample contains said plurality of calibrators at different known concentration levels and at least some of these analytical samples do not contain at least one of said calibrators, wherein said mass analysing obtains an ion signal for the analyte of interest in each of these analytical samples, and wherein said mass analysing obtains an ion signal for each known concentration level of said calibrators in the quality control sample in said further set of samples; b) updating said calibration relationship, or determining a new calibration relationship, using the ion signal obtained for each known concentration level of said calibrators in step a); and c) determining the concentration or quantity of the analyte of interest in at least one analytical sample using the ion signal detected for the analyte of interest in that analytical sample and the updated or new calibration relationship. Different sets of samples may include quality control samples having different known concentrations or quantities of the analyte of interest therein. The method may be performed by automated machinery that: (i) receives analytical samples; (ii) adds said plurality of different known concentration levels of the calibrators to a quality control sample; (iii) selects a set of samples that includes the analytical samples and the quality control sample; and (iv) transfers the set of samples to one or more mass spectrometer so as to then perform a method according to a second aspect of the present invention. Each of the analytical samples that is received at the automated machinery may have an identifier thereon that represents the type of sample that it is and / or a test that is required to be performed on that sample. The automated machinery may read the identifier and select analytical samples of the same type and / or that are to undergo the same test and performs the method described above on a set of samples that contains these analytical samples. Although embodiments have been described in which the samples are mass analysed, it is contemplated that the samples may be analysed using other techniques. Accordingly, from a third aspect the present invention provides a method of determining the concentration or quantity of an analyte of interest in an analytical sample comprising: analysing a first set of analytical samples, wherein one of the analytical samples contains a plurality of calibrators at different known concentration levels and another of the analytical samples does not contain at least one of said calibrators, wherein said analysing obtains a signal for an analyte of interest in each of the analytical samples, and wherein said analysing obtains a signal for each known concentration level of said calibrators in said one of the analytical samples; determining a calibration relationship based on said plurality of different known concentration levels of the calibrators and their respective signals obtained from analysing said one of the analytical samples; and determining the concentration or quantity of the analyte of interest in at least one of said set of analytical samples using the signal detected for the analyte of interest in that analytical sample and the calibration relationship. The third aspect of the present invention may have any of the features described in relation to the first aspect of the present invention, except that the mass analysis steps described need not be mass analysis steps and may instead be performed by other types of analysis. For example, in the third aspect of the invention said analysing may comprise ion mobility analysing. In such ion mobility analysing each sample is ionised and the resulting ions, or ions derived therefrom, are separated according to their ion mobility. The separated ions are then detected at an ion detector such that ions having different ion mobilities generate ion signals at different times. Therefore, different ion signals may be obtained for the analyte of interest and for the calibrators. Accordingly, said analysing may comprise ion mobility analysing the first set of analytical samples, wherein said ion mobility analysing obtains an ion signal for an analyte of interest in each of the analytical samples, and wherein said ion mobility analysing obtains an ion signal for each known concentration level of said calibrators in said one of the analytical samples. The calibration relationship may be determined based on said plurality of different known concentration levels of the calibrators and their respective ion signals obtained from ion mobility analysing said one of the analytical samples. The concentration or quantity of the analyte of interest in at least one of said set of analytical samples may be determined using the ion signal detected for the analyte of interest in that analytical sample and the calibration relationship. Alternatively, the samples may be analysed by techniques other than ion mobility analysis, such as optical analysis (e.g. spectroscopy). A fourth aspect the present invention provides a method of determining the concentration or quantity of an analyte of interest in an analytical sample comprising: analysing a first set of samples that comprises a quality control sample and a plurality of analytical samples, wherein said quality control sample contains a plurality of calibrators at different known concentration levels and at least some of the analytical samples do not contain at least one of said calibrators, wherein said analysing obtains a signal for an analyte of interest in each of the analytical samples, and wherein said analysing obtains a signal for each known concentration level of said calibrators in said quality control sample; determining a calibration relationship based on said plurality of different known concentration levels of the calibrators and their respective ion signals obtained from analysing said quality control sample; and determining the concentration or quantity of the analyte of interest in at least one of said analytical samples using the signal detected for the analyte of interest in that analytical sample and the calibration relationship. The fourth aspect of the present invention may have any of the features described in relation to the second aspect of the present invention, except that the mass analysis steps described need not be mass analysis steps and may instead be performed by other types of analysis. For example, in the fourth aspect of the invention said analysing may comprise ion mobility analysing. In such ion mobility analysing each sample is ionised and the resulting ions, or ions derived therefrom, are separated according to their ion mobility. The separated ions are then detected at an ion detector such that ions having different ion mobilities generate ion signals at different times. Therefore, different ion signals may be obtained for the analyte of interest and for the calibrators. Accordingly, said analysing may comprise ion mobility analysing the first set of samples, wherein said ion mobility analysing obtains an ion signal for an analyte of interest in each of the analytical samples, and wherein said ion mobility analysing obtains an ion signal for each known concentration level of said calibrators in said quality control sample. The calibration relationship maybe determined based on said plurality of different known concentration levels of the calibrators and their respective ion signals obtained from ion mobility analysing said quality control sample. The concentration or quantity of the analyte of interest in at least one of said analytical samples maybe determined using the ion signal detected for the analyte of interest in that analytical sample and the calibration relationship. Alternatively, the samples may be analysed by techniques other than ion mobility analysis, such as optical analysis (e.g. spectroscopy). The present invention also provides an apparatus that is arranged and configured to perform any of the methods described herein. Accordingly, the present invention provides automated apparatus having control circuitry and being configured to perform any of the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS Various embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which: Fig. 1 shows a layout of a typical batch of samples on a 96-well sample plate; Fig. 2 illustrates the concentrations of calibrator and analyte in different samples using a conventional approach; Fig. 3 shows a 96-well sample plate according to an embodiment of the present invention; Fig. 4 illustrates the concentrations of calibrators and analyte of interest in different samples according to the embodiment of Fig. 3; Figs. 5A-5D show example ion-chromatograms for an analyte of interest and three different calibrators; Fig. 6 shows a calibration relationship that is calculated based on the peak areas measured from the ion chromatograms shown in Figs. 5B-5D; Fig. 7 shows a 96-well sample plate according to another embodiment of the present invention in which the calibrators are provided in the first and last analytical samples and also in an analytical sample that is approximately mid-way between the first and last analytical samples; Fig. 8 illustrates the concentrations of calibrators and analyte of interest in different samples according to the embodiment of Fig. 7; Fig. 9 shows a 96-well sample plate according to another embodiment of the present invention in which the calibrators are provided in every 6th analytical sample; Fig. 10 illustrates the concentrations of calibrators and analyte of interest in different samples according to the embodiment of Fig. 9; Fig. 11 shows a 96-well sample plate according to another embodiment of the present invention in which the calibrators are provided in quality control samples; and Fig. 12 shows an embodiment of automated machinery used in performing the methods described herein. DETAILED DESCRIPTION Liquid chromatography mass spectrometry (LC-MS) is regularly used within a clinical laboratory setting to determine concentrations of various clinically relevant molecules in a biological patient sample, such as blood, blood plasma, blood serum, urine, stool, saliva or cerebrospinal fluid (CSF). For example, it may be desired to quantify the amount of one or more certain analytes in the sample, such as steroid hormones, vitamins, drugs of abuse (for example opiates), and therapeutic drugs such as immunosuppressants, antiepileptics, antipsychotics, antifungals, and anti-infective drugs. There is also a growing interest in peptide and protein analyses as potential indicators of disease or disorder. LC-MS is often undertaken in a batch-based workflow, i.e. multiple samples are processed simultaneously, with each sample in the batch undergoing the same preparation and analysis processes. For example, a 96-well sample plate is typically loaded with multiple samples in multiple respective sample wells, and then these samples are analysed in the same way. For instance, the samples may be analysed so as to test for a panel (i.e. plurality) of analytes of interest or for a single analyte of interest. It is important to standardise the results of the analyses and to ensure matrix effects are calibrated. Matrix effects occur when one or more compound in the matrix results in the ion signal detected for an analyte of interest being suppressed or enhanced relative to what it would be if that one or more compound was not present. In order to standardise the results of the analyses and to ensure matrix effects are calibrated, good laboratory practice (for example, CLSI C62A or equivalent guidelines) suggests that the following types of samples should be included in the batch being analysed:- an analytical sample, a solvent blank sample, a matrix blank sample, a matrix double blank sample, quality control samples, a blinded or challenge quality control sample, and calibrator samples. These types of sample are described in more detail below. It will be appreciated that the above list of sample types is not exhaustive and there may also be more specialist sample types that are used. It should also be appreciated that, subject to the proper considerations and validation, not all of the above sample types need be used in all cases. An analytical sample is a sample, generally from a patient, which contains an unknown quantity of one or more analyte of interest that is desired to be measured. Typically, each analytical sample will be mixed with a known quantity of an internal standard. An internal standard is a compound that is added to an analytical sample at a known concentration. The internal standard and analyte of interest may then both be measured and the response of the analyte measurement adjusted based on the measurement response of the internal standard. This may be considered to be a single point temporary calibration adjustment of the analyte measurement. It is required that the internal standard is distinguishable from the analyte of interest by the mass spectrometer and also that it behaves, chemically and physically, in as similar manner as possible to the analyte of interest during storage, measurement and analysis. As such, the internal standard is often a compound that is the same as the analyte of interest, except that one or more of its atoms has been substituted for a different, stable isotope of that atom. Such an internal standard is frequently known as a stable isotope labelled internal standard (SILS). In other words, the internal standard is often an analogue of the analyte of interest, labelled with a stable isotope to produce a compound that is substantially chemically identical but which has a different molecular mass. As such, any change in the measurement response of the internal standard due to the processing and analysing conditions would also be expected to occur for the analyte of interest. The internal standard may also be used as a form of risk control for the analysis, e.g. to detect where a sample might have been omitted during a manual sample preparation, as no internal standard would be detected. A solvent blank is a sample of the solvent that is used to carry the analytical sample. The analysis of the solvent blank sample is useful for determining if there is contamination in the analytical system. Typically, the solvent blank sample is analysed first when analysing the batch of samples. A matrix blank sample is a sample that is representative of the matrix present in the analytical sample (e.g. whole blood, blood plasma, blood serum, urine etc.) and which has the requisite internal standard added to it. In each batch that is analysed there may be multiple matrix blank samples. The analysis of a matrix blank sample is used to check for unintentional contamination of the batch with analyte of interest from a source other than the analytical sample. The matrix blank sample is sometimes referred to as a blank quality control sample (or QC-0). A double matrix blank sample is a sample that is representative of the matrix present in the analytical sample, but to which no internal standards or analyte of interest have been added. The analysis of the double matrix blank sample is used to check whether the source matrices used to run the analysis are contaminated with either analyte or internal standards. A quality control sample is a sample that contains an amount of analyte of interest at a known concentration. These samples are used to check that the calibration of the instrument performing the analysis is still valid and to determine the accuracy of the quantity or concentration of analyte of interest determined by the system. In other words, the analyte in the quality control sample is analysed and the resulting signal converted into a quantity or concentration of the analyte using the calibration. If this concentration matches the known concentration, within a predetermined tolerance, then the calibration is determined to be valid. On the other hand, if the determined concentration does not match the known concentration, and is outside of the predetermined tolerance, then the calibration is determined not to be valid. Typically, a quality control sample is measured both before and after the analytical samples in the batch are measured, so as to determine that there has been no change in response of the system in the meantime, e.g. due to contamination during the analysis. As mentioned above, the determined quantity or concentration of the analyte must typically be within the stated error of the measurement, although one measurement outside the measurement error window may be allowable. Quality control samples are frequently provided having low, high and medium known concentrations of the analyte. A blinded or challenge quality control sample is a sample that is submitted externally by an auditing body (either internal or external to the laboratory). This type of sample is submitted for analysis in order to check the laboratory practice and the quality of results from the analysis. Typically, the laboratory or operator is unaware that a sample is a blinded or challenge quality control sample and it is handled identically to an analytical sample. Calibrator samples are a plurality of samples that are used to obtain the calibration relationship between a detected analyte signal and the quantity or concentration of that analyte. Each of these calibrator samples is representative of the matrix present in the analytical samples and includes the internal standard added to it at a known concentration. Each of the calibrator samples also has a known concentration of the analyte in it, wherein different calibrator samples have different known concentrations of the analyte in it. The different calibrator samples are typically contained within separate sample wells. The calibrator samples are analysed and, as the concentrations of the analyte in each of the calibrator samples is known, the measured signal from each of these samples is able to be correlated with the concentration of analyte giving rise to it. This allows inference of the quantity of analyte present in a sample based of the response of the system. It is often important that calibrator samples contain the same matrix as the analytical samples so as to ensure that any matrix suppression or enhancement effects are accounted for in the measurement. Fig. 1 shows a layout of a typical LC-MS batch of samples on a 96-well sample plate. The sample plate has 8 rows (labelled A-H) of sample wells, where each row has 12 sample wells (labelled 1-12). In the example shown in Fig. 1, the sample plate includes a solvent blank sample at sample well A1, a double matrix blank sample at sample well A2, a matrix blank sample at sample well A3, and six calibrator samples at sample wells A4-A9. The six calibrator samples have six different respective known concentrations of the analyte, which are labelled as calibrator levels 1-6 in the diagram. The sample plate also includes a matrix blank sample at sample well A10, a quality control sample having a first relatively low known concentration of the analyte at sample well A11, a quality control sample having a second higher known concentration of the analyte at sample well A12, and a quality control sample having a third still higher known concentration of the analyte at sample well B1. The sample plate also contains a quality control sample having the third (highest) known concentration of the analyte at each of sample wells E3 and H12. The sample plate also contains a quality control sample having the first (lowest) known concentration of the analyte at each of sample wells C7 and H10. The sample plate also contains a quality control sample having the second (middle) known concentration of the analyte at each of sample wells F12 and H11. The sample plate also contains a matrix blank sample at sample well H9. All of the remaining sample wells in the sample plate are filled with analytical samples. In use the samples may be analysed in sequence, starting with the first row and then moving to the next row until all samples on the plate have been analysed. For example, the method may first sequentially analyse consecutive samples in the first row (i.e. samples A1 to A12), and then sequentially analyse consecutive samples in the second row (i.e. samples B1 to B12) etc. Accordingly, the blank samples in sample wells A1-A3 are analysed sequentially and then the calibrator samples in sample wells A4-A9 are analysed sequentially. The signals detected from the calibrator samples, and their known concentrations of analyte, are then used to determine a calibration curve that correlates a measured analyte signal to the concentration of that analyte. The samples in the remaining sample wells are then mass analysed. The calibration curve is used to convert the response measured for the analyte in any given analytical sample to a quantity or concentration value of the analyte in that sample. The quantity or concentration value for the analyte in any given analytical sample may be adjusted using the measured response of the internal standard in that analytical sample. The quality control samples are analysed and used for their purpose as has been described above. Fig. 2 illustrates the known concentrations of the analyte in the calibrator samples relative to the expected range of unknown concentrations of the analyte in some of the analytical samples. This illustration depicts the quantity (amount) of analyte in a sample for different samples to be analysed. In this example seven calibrator samples having different known concentrations of the analyte are provided in seven different respective sample wells, which are represented by the values at times 1-7 on the x-axis. Analytical samples having unknown concentrations of the analyte are provided in other sample wells, which are represented by the values at times 8-12 on the x-axis. The vertically elongated rectangle at each of times 8-12 represents the range of concentrations of analyte in an analytical sample that might be expected. The calibrator samples are selected to have concentrations that extend from a concentration below the minimum expected concentration to a concentration above the maximum expected concentration, with concentrations evenly spaced therebetween. The above described calibration process is undertaken for each batch in conventional LC-MS workflows. This is because current LC-MS workflows are highly flexible, with the same LC-MS system open to be used in many different ways. For example, the same system may be used to detect multiple different analytes or different panels of analytes, it may be used in different methods such as to analyse samples having different sample preparation steps, it may use different chromatography columns, different chromatography solvents, different chromatography washes, and different chromatographic gradients etc. Due to the variety of methods that may be used on the same system, good laboratory practice recommends the acquisition of fresh calibration samples at the start of each batch of samples to be analysed. This may change, however, in cases where multiple small batches of samples might be run sequentially, or when a system is used continuously without configuration change. In any event, it is desirable to analyse calibration samples as infrequently as possible while retaining the desired level of quality of analysis. It is also desirable to minimise the number of sample wells required to hold calibrator samples, particularly for batches that contain relatively few samples. It is also desirable to reduce as much as possible the time taken to analyse calibrator samples so as to improve sample throughput and reduce the time taken to release a result. It is also desirable to reduce as far as reasonably possible the number of calibrators or standards employed across the full batch. Fig. 3 shows a 96-well sample plate according to an embodiment of the present invention. The sample plate is filled with samples in a similar manner to that shown in Fig. 1, except that calibrator samples are not provided separately in their own sample wells. Instead, calibrators having different known concentrations are provided in one of the analytical samples for use in forming a calibration relationship relating the ion signal detected by the mass spectrometer when analysing a sample to the quantity or concentration of analyte in that sample, as will be discussed further below. In the example described below, six calibrators having six different known concentrations are provided in the same sample, although it will be appreciated that fewer or a greater number of calibrators may be used in each analytical sample. The calibrators should ideally behave, chemically, in as similar manner as possible to the analyte of interest. As such, the each of the calibrator may be a compound that is the same as the analyte of interest, except that one of more of the atoms in its chemical structure has been substituted for a different, stable isotope of that atom. Such a calibrator will be referred to herein as a stable isotope labelled calibrator (SILC). As such, in the embodiment having six calibrators of different known concentrations in the same analytical sample, these six calibrators will be six different SILCs having six different respective molecular masses. In the following embodiments the calibrators will be described as SILCs, although it will be appreciated that other types of calibrator may be used instead, such as compounds are different to the analyte of interest but in the same family of compounds as the analyte of interest. In the example shown in Fig. 3, the sample plate has 8 rows (labelled A-H) of sample wells, where each row has 12 sample wells (labelled 1-12). The sample plate includes a solvent blank sample at sample well A1, a double matrix blank sample at sample well A2, a matrix blank sample at sample well A3, a quality control sample having a first relatively low known concentration of the analyte at sample well A4, a quality control sample having a second higher known concentration of the analyte at sample well A5, and a quality control sample having a third still higher known concentration of the analyte at sample well A6. An analytical sample having the six SILCs of different concentrations is provided at sample well E1, i.e. approximately half way along the sample plate. A quality control sample having the first relatively low known concentration of the analyte is provided at sample wells C7 and H10, a quality control sample having the third known concentration of the analyte is provided at sample wells E3 and H12, and a quality control sample having the second known concentration of the analyte is provided at sample wells F12 and H11. A sample blank is provided at sample well H9. All of the remaining sample wells are filled with analytical samples that do not contain any SILCs, i.e. sample wells A7-C6, C8-D12, E2, E4-F11, andG1-H8. An internal standard may also be added to each of the analytical samples in the batch, where the internal standard has the same concentration in all of the analytical samples. The internal standard should also ideally behave chemically in as similar manner as possible to the analyte of interest. As such, the internal standard may be a compound that is the same as the analyte of interest, except that one of more of the atoms in its chemical structure has been substituted for a different, stable isotope of that atom. Such an internal standard compound will be referred to as a stable isotope labelled internal standard (SILIS) compound. In the following embodiments the internal standard compound will be described as a SILIS compound, although other types of internal standard could be used, such as a compound that is different to the analyte of interest but in the same family of compounds as the analyte of interest. The internal standard compound and the calibrators may be different, at least in the sense that they are distinguishable by the mass spectrometer. For example, the SILIS compound and the SILCs may be the same chemical compound, but wherein one or more atoms in each of the SILCs is a different isotope to in the SILIS. It is alternatively contemplated that the same concentration of one of the SILCs may be added to all of the analytical standards so as to be used as the internal standard. In use the samples on the sample plate were analysed one at a time and consecutively by LC-MS in the sequence described above, i.e. from left to right in each row and one row at a time, starting with row A and then proceeding to the next row consecutively from row B to row H. As will be appreciated, in LC-MS a sample is introduced into a liquid chromatograph device that separates components in the sample such that they elute from the liquid chromatograph device over different time periods. As the components elute they are ionised by an ionisation source and the resulting ions are mass analysed in the mass spectrometer. In this example the mass spectrometer is a tandem (MS / MS) mass spectrometer that monitors certain multiple reaction monitoring (MRM) transitions and produces an ion chromatogram for each of these, i.e. records the detected ion signal as a function of time. The method starts by using LC-MS to sequentially analyse the solvent blank sample, double blank sample, and quality control samples from sample wells A1-A6. The method then proceeds to sequentially analyse the samples in the subsequent sample wells, wherein for each of the analytical samples the mass spectrometer monitors different MRM transitions in order to detect an ion signal for the analyte of interest, and the SILIS (if one has been added to the analytical samples). When the mass spectrometer mass analyses the analytical sample that contains the SILCs, the mass spectrometer monitors an MRM transition in order to detect an ion signal for the analyte of interest, and also monitors six different MRM transitions in order to detect an ion signal for each of the SILCs. If a separate SILIS has also been added to the analytical samples then the mass spectrometer also monitors an MRM transition in order to detect an ion signal for the SILIS. As one skilled in the art will appreciate, in order to monitor any given MRM transition the mass spectrometer controls a first mass filter to have a mass transmission window such that it is only capable of transmitting precursor ions having a specific mass to charge ratio. The precursor ions that are transmitted by the first mass filter are guided into a fragmentation or reaction device, in which the precursor ions are fragmented or reacted so as to form fragment or product ion species. The fragment or product ions are then transmitted to a second mass filter, which is operated so as to have a mass transmission window such that it is only capable of transmitting ions having a specific mass to charge ratio to an ion detector. The mass to charge ratio values of the mass transmission windows for the first and second mass filters are selected so that if ions are detected by the ion detector then it is determined that ions of interest are present in the sample being analysed. The ion signal is also used to determine the quantity of ions in the sample. For example, for the MRM transition for the analyte of interest, the mass to charge ratio values of the mass transmission windows for the first and second mass filters are selected so that only ions having a mass to charge ratio corresponding to a specific fragment ion species of the analyte of interest are able to be detected by the ion detector. The ion signal for these ions may therefore be used to determine the quantity or concentration of the analyte of interest in the sample. A different MRM transition for the analyte of interest may also be monitored, wherein the mass to charge ratio values of the mass transmission windows for the first and second mass filters are selected so that only ions having a mass to charge ratio corresponding to a different fragment ion species of the analyte of interest are able to be detected by the ion detector. The mass spectrometer may monitor this MRM transition so that if ions are detected for this MRM transition then it is confirmed that it is actually the analyte of interest that is being detected, since it is highly unlikely that a precursor ion other than the analyte of interest would give rise to ion signals for both MRM transitions. Similarly, for each SILC, the mass to charge ratio values of the mass transmission windows for the first and second mass filters are selected so that only ions having a mass to charge ratio corresponding to a specific fragment ion species of the SILC are able to be detected by the ion detector. The ion signal for these ions may therefore be used to determine the quantity or concentration of the SILC in the sample. A different MRM transition for the SILC may also be monitored, wherein the mass to charge ratio values of the mass transmission windows for the first and second mass filters are selected so that only ions having a mass to charge ratio corresponding to a different fragment ion species of the SILC are able to be detected by the ion detector. The mass spectrometer may monitor this MRM transition so that if ions are detected for this MRM transition then it is confirmed that it is actually the SILC that is being detected, since it is highly unlikely that a precursor ion other than the SILC would give rise to ion signals for both MRM transitions. For the SILIS, the mass to charge ratio values of the mass transmission windows for the first and second mass filters are selected so that only ions having a mass to charge ratio corresponding to a specific fragment ion species of the SILIS are able to be detected by the ion detector. The ion signal for these ions may therefore be used to determine the quantity or concentration of the SILIS in the sample. A different MRM transition for the SILIS may also be monitored, wherein the mass to charge ratio values of the mass transmission windows for the first and second mass filters are selected so that only ions having a mass to charge ratio corresponding to a different fragment ion species of the SILIS are able to be detected by the ion detector. The mass spectrometer may monitor this MRM transition so that if ions are detected for this MRM transition then it is confirmed that it is actually the SILIS that is being detected, since it is highly unlikely that a precursor ion other than the SILIS would give rise to ion signals for both MRM transitions. Preferably, the MRM transition(s) for each of the analyte of interest, the SILIS, and the SILCs are selected such that ions will be detected at the detector if these undergo the same manner of fragmentation, i.e. these compounds are preferably fragmented in the same place(s) of the chemical structure. The MRM transition(s) for each of the analyte of interest, the SILIS, and the SILCs will however require the mass filters to have slightly different mass transmission windows, as these compounds have atoms of different isotopes in them. This is how the mass spectrometer is able to distinguish them from each other when they are present in the same sample. For the analysis of each of the samples, the mass spectrometer may cycle through the above-described MRM transitions as sample elutes from the liquid chromatograph device, so as to obtain ion chromatograms for each of the MRM transitions that correspond to the analyte of interest, the SILIS, and the SILCs. The mass spectrometer may be programmed not to monitor the MRM transitions for the SILCs when mass analysing samples that are known not to contain the SILCs. The mass spectrometer determines the abundance of ions in the ion peak for each ion chromatogram, e.g. by determining the area under each peak. The mass spectrometer may have slightly different responses when detecting the analyte of interest, the SILIS, and the SILCs and as such a relative response factor may be calculated for each of the analyte of interest and the SILCs (relative to the SILIS compound). For example, the abundance of the analyte of interest may be divided by the abundance of the SILIS so as to obtain a relative response factor for the analyte of interest, and similarly the abundance of each SILC may be divided by the abundance of the SILIS so as to obtain a relative response factor for the SILC compound. The relative response factor for each SILC may then be used to calculate an apparent concentration for the SILC, e.g. by multiplying the known concentration of that SILC by its relative response factor. As such, the mass spectrometer determines a data pair consisting of the ion signal detected (ion abundance) and the apparent quantity or concentration of the SILC that gave rise to this signal. This process is performed on each SILC so as to obtain a data pair for each of the six concentrations of the SILCs. These six data pairs are then used to form a calibration relationship between an ion signal that is detected and the quantity or concentration of compound giving rise to the detected ion signal, e.g. by least squares fitting of the instrument response to the known concentrations (or quantities). The mass spectrometer then calculates the quantity or concentration of the analyte of interest in each analytical sample, and optionally the quality control samples, using the ion signal detected for the analyte of interest in any given one of these samples and the calibration relationship that has been determined. Fig. 4 illustrates the known concentrations of the SILCs relative to the expected range of concentrations of the analyte of interest in some of the samples on the sample plate of Fig. 3. More specifically, concentrations are shown for sample numbers 46-52, which correspond to sample wells D10-E4 on the sample plate. The vertically elongated rectangles represent the range of concentrations of the analyte of interest in the analytical samples that might be expected. The concentration of the analyte of interest in sample number 51, which corresponds to sample well E3, is expected to differ from the others shown as it contains the quality control sample having the third (highest) concentration of the analyte. It can be seen that the known concentrations of the SILCs are selected so as to extend over a range from a concentration that is below the minimum expected concentration of the analyte of interest to a concentration that is above the maximum expected concentration of the analyte of interest, with the remaining known concentrations of the SILCs relatively evenly spaced therebetween. However, it will be appreciated that the known concentrations of the SILCs need not be below the minimum expected concentration of the analyte of interest and / or above the maximum expected concentration of the analyte of interest. Although the analysis of a single batch of samples has been described, i.e. the batch of samples on the sample plate shown, it will be appreciated that one or more further batches of samples may also be analysed in a corresponding manner. These further batches may be prepared in the same way as has been described above, i.e. by adding the SILCs to the analytical samples in the same way as has been described above. When the method transitions from analysing the last analytical sample in one batch to the first analytical sample in the new batch, the method may continue to use the calibration relationship that was last determined at the end of the analysis of the last batch and then use the data pairs obtained from the new batch to update that calibration relationship. Alternatively, the calibration process may begin afresh for the next batch of samples. As mentioned above, although a set of six SILCs having six different concentrations has been described as being added to one of the analytical samples, the number of SILCs having different concentrations that are added to the analytical sample may be different to this. An example will now be described that models an embodiment in which the analyte of interest having an unknown concentration (e.g. in a patient sample) is testosterone and in which there are three SILCs having different concentrations present in the analytical sample. In this example, the SILCs are di-, tri-, and penta-deuterated testosterone. During the analyses described below, the analyte of interest of unknown concentration is monitored using an MRM transition of 289.25 >96.9. The di-deuterated testosterone is monitored using an MRM transition of 291.25 >98.9. The tri-deuterated testosterone is monitored using an MRM transition of 292.25 >96.9. The penta-deuterated testosterone is monitored using an MRM transition of 294.25 >99.9. It will therefore be appreciated that when the analytical sample containing the SILCs is analysed the mass spectrometer will monitor the MRM transition for the analyte of interest and also the MRM transitions for each of di-, tri- and penta-deuterated testosterone, and produce ion chromatograms for these MRM transitions. Figs. 5A-5D show example ion-chromatograms for the above described MRM transitions. More specifically, Fig. 5A shows an ion-chromatogram for the analysis of the analyte of interest having an unknown concentration, which was obtained by monitoring the MRM transition of 289.25 >96.9. Fig. 5B shows an ion-chromatogram for the analysis of 0.2 ng / mL di-deuterated testosterone, which was obtained by monitoring the MRM transition of 291.25 >98.9. Fig. 5C shows an ion-chromatogram for the analysis of 4.0 ng / mL tri-deuterated testosterone that was obtained by monitoring the MRM transition of 292.25 >96.9. Fig. 5D shows an ion-chromatogram for the analysis of 10.0 ng / mL penta-deuterated testosterone that was obtained by monitoring the MRM transition of 294.25 >99.9. The integrated peak area is determined for the peak in each ion chromatogram, so as to obtain a data pair consisting of the peak area and known concentration for each of the SILCs. These data pairs for the SILCs are then used to form a calibration relationship that relates a peak area measured in an ion chromatogram to a concentration of analyte of interest. Fig. 6 shows a calibration relationship that is calculated based on the peak areas measured from the ion chromatograms shown in Figs. 5B-5D and the known concentrations of the SILCs that gave rise to these peaks. The peak area for the analyte of interest that is shown in the ion chromatogram of Fig. 5A was determined and, using the calibration relationship shown in Fig. 6, it was determined that the analyte of interest has a concentration of 2.85 ng / mL. Fig. 7 shows a 96-well sample plate according to another embodiment of the present invention. This embodiment is the same as that shown in Fig. 3, except that the set of six SILCs are also added to sample wells C7 and H8. As such, this embodiment is able to generate additional calibration relationships at the start and end of the analysis sequence. In use the samples on the sample plate are analysed as has been described above in relation to Fig. 3, except that because the SILCs are provided in three samples in the embodiment shown in Fig. 7, the calibration relationship is determined three times. After the analytical sample at sample well A7 has been mass analysed, the mass spectrometer is able to determine a calibration relationship based on the known concentration levels of SILCs1-6, in a corresponding manner to that described in the above embodiments. The previously analysed samples in the batch, i.e. the quality control samples, may then be calibrated using this relationship. The method then proceeds to sequentially analyse the further samples in other sample wells of the sample plate. For example, the method will next sequentially analyse the samples from sample wells A8-D12 in a corresponding manner to that described above. The method may use the calibration relationship determined using the SILCs in sample well A7 to calibrate the concentration or quantity of the analyte of interest in some or all of the samples in sample wells A8-D12. The mass spectrometer then mass analyses the sample in sample well E1 and determines a new calibration relationship based only on the known concentration levels of SILCs1-6 therein (i.e. not based on the SILCs in sample well A7), in a corresponding manner to that described above. The method may use the calibration relationship determined using the SILCs in sample well E1 to calibrate the concentration or quantity of the analyte of interest in some of the samples in sample wells A8-D12. For example, the calibration relationship obtained from the SILCs in sample well A7 may be used to calibrate the concentration or quantity of the analyte of interest in a first set of samples that were consecutively analysed (between samples A7 and E1) and the calibration relationship obtained from the SILCs in sample well E1 may be used to calibrate the concentration or quantity of the analyte of interest in a second different set of samples that were consecutively analysed (between samples A7 and E1), wherein the first set were analysed during a time period closer to the analysis of the sample in sample well A7 than the second set. The method then proceeds to sequentially analyse the further samples in other sample wells of the sample plate. For example, the method will next sequentially analyse the samples from sample wells E2-H12 in a corresponding manner to that described above. The method may use the calibration relationship determined using the SILCs in sample well E1 to calibrate the concentration or quantity of the analyte of interest in some or all of the samples in sample wells E2-H8 (and H9-12). The mass spectrometer mass analyses the sample in sample well H8 and determines a new calibration relationship based only on the known concentration levels of SILCs1-6 therein (i.e. not based on the SILCs in sample well A7 or E1), in a corresponding manner to that described above. The method may use the calibration relationship determined using the SILCs in sample well H8 to calibrate the concentration or quantity of the analyte of interest in some of the samples in sample wells E2-H8 (and H9-12). For example, the calibration relationship obtained from the SILCs in sample well E1 may be used to calibrate the concentration or quantity of the analyte of interest in a third set of samples that were consecutively analysed (between samples E1 and H8) and the calibration relationship obtained from the SILCs in sample well H8 may be used to calibrate the concentration or quantity of the analyte of interest in a fourth different set of samples that were consecutively analysed (between samples E1 and H8), wherein the third set were analysed during a time period closer to the analysis of the sample in sample well E1 than the fourth set. In the above embodiment, different sets of SILCs in the different sample wells are used to form different calibration relationships that are applied to separate sets of samples. However, alternatively, the calibration relationship determined using the SILCs from one sample well may be updated based on the mass analysis of the SILCs from another sample well. For example, a calibration relationship may be determined using the SILCs from sample well A7, as described above. This involves determining a data pair consisting of the ion signal detected (ion abundance) and the concentration or quantity of the SILC compound that gave rise to this signal for each of the SILCs, and then using these data pairs to form a calibration relationship. When the mass spectrometer mass analyses the sample in sample well E1 it may determine a data pair consisting of the ion signal detected (ion abundance) and the concentration or quantity of the SILC compound that gave rise to this signal for each of the SILCs in that sample well. Each of these data pairs may be used to update the previously calculated calibration relationship, e.g. by inclusion of the subsequently obtained data pairs in the least squares fitting process described above. The updated calibration relationship may be used to calibrate the concentration or quantity of the analyte of interest in at least some of the samples that were previously analysed and / or that will be subsequently analysed. When the mass spectrometer mass analyses the sample in sample well H8 it may determine a data pair consisting of the ion signal detected (ion abundance) and the concentration or quantity of the SILC compound that gave rise to this signal for each of the SILCs in that sample well. These data pairs may be used to update the previously updated calibration relationship, e.g. by inclusion these data pair in the least squares fitting process described above. The updated calibration relationship may be used to calibrate the concentration or quantity of the analyte of interest in at least some of the samples previously analysed. Fig. 8 illustrates the known concentrations of the SILCs relative to the expected range of concentrations of the analyte of interest in some of the samples on the sample plate of Fig. 7. More specifically, concentrations are shown for sample numbers 7-8, 48-50 and 90-91, which correspond to sample wells A7-A8, D12-E2 and H7-H8, respectively. The vertically elongated rectangles represents the range of concentrations of the analyte of interest in the analytical samples that might be expected. It can be seen that the known concentrations of the SILCs are selected so as to extend over a range from a concentration that is below the minimum expected concentration of the analyte of interest to a concentration that is above the maximum expected concentration of the analyte of interest, with the remaining known concentrations of the SILCs relatively evenly spaced therebetween. However, it will be appreciated that the known concentrations of the SILCs need not be below the minimum expected concentration of the analyte of interest and / or above the maximum expected concentration of the analyte of interest. Fig. 9 shows a 96-well sample plate according to another embodiment of the present invention. This embodiment is the same as that shown in Fig. 7, except that the set of six SILCs are added to every nth analytical sample. The value of n may be the same as the number of SILCs that are provided in each sample that has SILCs (i.e. 6 in this embodiment), although this need not be the case and n could be a higher or lower integer. As can be seen, samples other than analytical samples may be analysed between analysing the analytical samples and these may not count towards the value n. The SILCs in each sample that contains them may be used to generate its own calibration relationship that is only applied to either the previous or subsequent n analytical samples. Alternatively, when a sample containing SILCs is mass analysed the resulting data for the SILCs may be used to update a calibration relationship that was previously determined based on the prior analysis of SILCs from a different sample. The calibration relationship may be updated using only data from the mass analysis of a predetermined number of samples containing SILCs (which may be fewer than the number of samples on the sample plate containing SILCs). The updated calibration relationship may be used to calibrate the concentration or quantity of the analyte of interest in some or all of the samples. Fig. 10 illustrates the known concentrations of the SILCs relative to the expected range of concentrations of the analyte of interest in some of the samples on the sample plate of Fig. 9. Fig. 11 shows a 96-well sample plate according to another embodiment of the present invention. This embodiment is the same as that shown in Fig. 3, except that the SILCs are not provided in any of the analytical samples and are instead provided in the quality control samples. The methods described herein may be performed by automated machinery that receives samples to be tested and selects some of them to be tested together, e.g. as shown in Fig. 12. Fig. 12 shows an embodiment of automated machinery that receives samples 2, e.g. which are deposited on a conveyer 3. The samples 2 have identifiers thereon, such as barcodes, that identify the type of sample that they are and / or the type(s) of test that they should be subjected to. The samples are conveyed to a reading device 4 that reads the information on the identifier. The automated machinery moves the samples from the conveyor 3 to a storage region 5, where samples of the same type and / or that are to be subjected to the same test(s) are grouped together in the storage region. Samples of different types and / or that are to be subjected to different tests (or different sets of tests) may be placed in the storage region in different groups. The automated machinery is configured to select a group of samples from the storage region 5 and prepare a batch of analytical samples from them, e.g. by transferring samples to sample wells on a sample plate 6 using a robotic arm 7 or other automated means. The automated machinery then performs sample preparation steps required in order to conduct the test(s) on the analytical samples, e.g. by adding one or more compound 8 to, or removing one or more compound from, the analytical samples. These steps are known to those skilled in the art and will not be described, save to say that the sample preparation steps involve adding the different known concentration levels of the SILCs to the analytical samples. The automated machinery then outputs the batch of prepared samples for mass analysis by the mass spectrometer. In the illustrated embodiment this is performed by moving the sample plate 6 to a conveyor 9, although other mechanism could be used. The automated machinery may include an automated sampler that automatically subjects the analytical samples to LC-MS as has been described above. Although the present invention has been described with reference to various embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims. For example, although embodiments have been described that use separate internal standards and SILCs, it will be appreciated that one or more of the SILCs could be used to perform the function of the internal standard. Although embodiments have been described in which a batch of 96 samples are provided on a sample plate, it will be appreciated that fewer or more samples may be provided in each batch and / or that they need not be provided on a sample plate. It is contemplated that the samples may not even be provided on sample plates or in batches, e.g. they may be provided as a stream of samples that are consecutively analysed. The samples on the sample plate have been described as being analysed consecutively, from left to right in each row and one row at a time, starting with row A and then proceeding to the next row. However, the samples may be analysed in any order. For example, the samples in each row may be analysed consecutively, from right to left. Alternatively, the samples may be analysed consecutively, from top to bottom in each column and one column at a time. The LC-MS analysis described herein may use an Ultra Performance Liquid Chromatography (UPLC) system, e.g. such as the Waters l-Class system. However, alternative approaches and chromatography pressure regimes may be used. The LC-MS analysis may use a single chromatography column and the samples may be injected into the column sequentially. Alternatively, different samples may be analysed during at least partially overlapping time periods by injecting them into different chromatography columns. The chromatography may be carried out according to any of the known techniques, such as multichannel chromatography, two-dimensional chromatography, flow injection analysis, or trap-and-elute chromatography etc.). Embodiments have been described in which the chromatographically separated samples are analysed by a tandem mass spectrometer, such as a triple quadrupole mass spectrometer, operating in a MRM mode. However, other mass analysis techniques and / or mass spectrometer may be used to obtain ion peaks for the analyte of interest and calibrators in any given sample. For example, a triple quadrupole mass spectrometer operating in a mode other than an MRM mode may be used. Alternatively, a single quadrupole mass spectrometer, time of flight mass spectrometer, Orbitrap mass spectrometer, FT-ICR mass spectrometer, or other mass spectrometer may be used. Although the samples have been described as being separated by liquid chromatography, the liquid samples need not be separated prior to analysis, or they may be separated by alternative techniques. For example, the samples may be subjected to capillary electrophoresis, infusion techniques or direct analysis prior to mass analysis. Although the samples have been described as being mass analysed, it is contemplated that the samples may additionally, or alternatively, be analysed using ion mobility analysis. Alternatively, the samples may be analysed by other techniques, such as optical analysis (e.g. spectroscopy). Various sample types and analytes of interest have been described herein. However, the invention is not limited to these. For example, the analyte of interest may be a steroid hormone, a vitamin, a drug of abuse (for example an opiate), or a therapeutic drug such as an immunosuppressant drug, an anti-epileptic drug, an anti-psychotic drug, an anti-infective drug, a steroid, or an anti-fungal drug etc. The analyte of interest may be an amino acid, a peptide or a protein. The methods described herein determine the concentration of an analyte of interest in the samples. However, it will be appreciated that the invention extends to determining the concentration of one or more additional analyte of interest in the analytical samples in a corresponding manner to that described. For example, each analyte of interest may have its own respective calibrators. The multiple analytes of interest may be in the same samples or may be in different respective samples. For instance, a given batch of samples to be analysed may contain a first set of analytical samples, and their associated quality control samples and / or blank samples, that are to be subjected to a first test (e.g. for an immunosuppressant drug panel) and a second different set of analytical samples, and their associated quality control samples and / or blank samples, that are to be subjected to a different test (e.g. for a steroid hormone panel).

Claims

1. A method of determining the concentration or quantity of an analyte of interest in an analytical sample comprising:mass analysing a first set of analytical samples, wherein one of the analytical samples contains a plurality of calibrators at different known concentration levels and another of the analytical samples does not contain at least one of said calibrators, wherein said mass analysing obtains an ion signal for an analyte of interest in each of the analytical samples, and wherein said mass analysing obtains an ion signal for each known concentration level of said calibrators in said one of the analytical samples;determining a calibration relationship based on said plurality of different known concentration levels of the calibrators and their respective ion signals obtained from mass analysing said one of the analytical samples; anddetermining the concentration or quantity of the analyte of interest in at least one of said set of analytical samples using the ion signal detected for the analyte of interest in that analytical sample and the calibration relationship.

2. The method of claim 1, wherein said set of analytical samples is formed from consecutively mass analysed analytical samples.

3. The method of claim 1 or 2, wherein said one of the analytical samples that contains the plurality of calibrators is either:i) the first analytical sample to be mass analysed in said set of analytical samples;ii) the final analytical sample to be mass analysed in said set of analytical samples; oriii) an analytical sample that is mass analysed substantially midway between the first and final analytical samples to be mass analysed in said set of analytical samples.

4. The method of claim 1, 2 or 3, wherein only a single analytical sample in said set of analytical samples contains all of said plurality of calibrators.

5. The method of claim 4, wherein the remaining samples in said set of analytical samples have none of said calibrators therein.

6. The method of claim 4, wherein the remaining samples in said set of analytical samples have only one of said calibrators therein, which is at the same concentration in all of said remaining samples.

7. The method of any preceding claim, comprising estimating a range of concentrations that the analyte of interest is expected to have in the analytical samples,wherein said different known concentration levels of the calibrators include a first concentration level above said range and / or a second concentration level below said range.

8. The method of any preceding claim, comprising estimating a range of concentrations that the analyte of interest is expected to have in the analytical samples, wherein said different known concentration levels of the calibrators includes at least one concentration level within said range.

9. The method of any preceding claim, wherein each of said calibrators is a compound that is the same as the analyte of interest, except that one of more of the atoms in its chemical structure has been substituted for a different, stable isotope of that atom.

10. The method of any preceding claim, further comprising performing a cycle that comprises:a) mass analysing a further set of analytical samples, wherein one of these analytical samples contains said plurality of calibrators at different known concentration levels and another of these analytical samples does not contain at least one of said calibrators, wherein this mass analysing obtains an ion signal for the analyte of interest in each of the analytical samples in the further set, and wherein the mass analysing obtains an ion signal for each known concentration level of said calibrators in said further set of the analytical samples;b) updating said calibration relationship, or determining a new calibration relationship, using the ion signal obtained for each known concentration level of said calibrators in step a); andc) determining the concentration or quantity of the analyte of interest in at least one analytical sample using the ion signal detected for the analyte of interest in that analytical sample and the updated or new calibration relationship.

11. The method of claim 10, wherein step c) comprises determining the concentration or quantity of the analyte of interest in at least one analytical sample in said further set of analytical samples using the ion signal detected for the analyte of interest in that analytical sample and the updated or new calibration relationship determined in step b).

12. The method of claim 10 or 11, comprising repeating said cycle a plurality of times.

13. The method of claim 12, wherein each time the cycle is performed, the updated ornew calibration relationship is used to determine the concentration or quantity of the analyte of interest in analytical samples that were mass analysed in step (a) of that cycle.

14. The method of claim 12 or 13, wherein each time the cycle is performed, step c) comprises determining the concentration or quantity of the analyte of interest in at leastone analytical sample in the previously mass analysed set of analytical samples using the ion signal detected for the analyte of interest in that analytical sample and the updated or new calibration relationship determined in step b).

15. The method of claim 12, 13 or 14, wherein the cycle is repeated for a pre-selected number of cycles during each of which step b) updates the calibration relationship obtained in the previous cycle, and then the cycle is performed a further time in which a new calibration relationship is determined that does not use the ion signals for the calibrants that were obtained in any preceding cycles.

16. The method of any one of claims 10-15, wherein all of the sets of analytical samples have the same number of analytical samples therein.

17. The method of any one of claims 10-16, wherein the number of analytical samples in each set of analytical samples is the same as the number of calibrators having different known concentration levels.

18. The method of any preceding claim, comprising providing a batch of samples containing analytical samples, wherein either:i) the only analytical samples in the batch are said first plurality of analytical samples; orii) the method comprises performing or repeating said cycle until all of the sets of analytical samples in the batch have been mass analysed.

19. The method of claim 18, wherein the batch of samples only includes said different known concentration levels of the calibrators in the first analytical sample that is mass analysed, in the final analytical sample that is mass analysed, and in a further analytical sample that is mass analysed between the first and final analytical samples; optionally wherein the further analytical sample is mass analysed substantially midway between mass analysing the first and final analytical samples.

20. The method of claim 18 or 19, comprising providing a plurality of batches of samples that include analytical samples and performing the method of any preceding claim on each of the batches, wherein either:(i) the different known concentration levels of the calibrators that are mass analysed first in any given batch are used to form a new calibration relationship that is used to determine the concentration or quantity of the analyte of interest in one or more analytical sample in that batch; or(ii) the different known concentration levels of the calibrants that are mass analysed first in any given batch are used to update the calibration relationship obtained at the end of analysing the previous batch, and the updated calibration relationship is used todetermine the concentration or quantity of the analyte of interest in one or more analytical sample in said given batch.

21. The method of any preceding claim, wherein the analytical samples are mass analysed using liquid chromatography mass spectrometry; optionally wherein each of the analytical samples is mass analysed using tandem mass spectrometry by monitoring an MRM transition for said analyte of interest, and at least the analytical samples containing the calibrators are mass analysed using tandem mass spectrometry by monitoring an MRM transition for each of the calibrators.

22. The method any preceding claim, wherein the number of calibrators having different known concentration levels is between four and seven calibrators.

23. The method of any preceding claim, wherein the method is performed by automated machinery that:(i) receives samples;(ii) adds said plurality of different known concentration levels of the calibrators to one of the analytical samples so as to form said set of analytical samples; and(iii) transfers the set of analytical samples to one or more mass spectrometer so as to then perform the method of any preceding claim.

24. The method of claim 23, wherein each of the analytical samples that is received at the automated machinery has an identifier thereon that represents the type of sample that it is and / or a test that is required to be performed on that sample; andwherein the automated machinery reads the identifier and selects analytical samples of the same type and / or that are to undergo the same test and performs step (ii) on these analytical samples.

25. The method of any preceding claim, wherein each analytical sample contains an unknown quantity or concentration of the analyte of interest, and / or wherein each analytical sample is not a quality control sample.

26. A method of determining the concentration or quantity of an analyte of interest in an analytical sample comprising:mass analysing a first set of samples that comprises a quality control sample and a plurality of analytical samples, wherein said quality control sample contains a plurality of calibrators at different known concentration levels and at least some of the analytical samples do not contain at least one of said calibrators, wherein said mass analysing obtains an ion signal for an analyte of interest in each of the analytical samples, and wherein said mass analysing obtains an ion signal for each known concentration level of said calibrators in said quality control sample;determining a calibration relationship based on said plurality of different known concentration levels of the calibrators and their respective ion signals obtained from mass analysing said quality control sample; anddetermining the concentration or quantity of the analyte of interest in at least one of said analytical samples using the ion signal detected for the analyte of interest in that analytical sample and the calibration relationship.

27. A method of determining the concentration or quantity of an analyte of interest in an analytical sample comprising:analysing a first set of analytical samples, wherein one of the analytical samples contains a plurality of calibrators at different known concentration levels and another of the analytical samples does not contain at least one of said calibrators, wherein said analysing obtains a signal for an analyte of interest in each of the analytical samples, and wherein said analysing obtains a signal for each known concentration level of said calibrators in said one of the analytical samples;determining a calibration relationship based on said plurality of different known concentration levels of the calibrators and their respective signals obtained from analysing said one of the analytical samples; anddetermining the concentration or quantity of the analyte of interest in at least one of said set of analytical samples using the signal detected for the analyte of interest in that analytical sample and the calibration relationship.

28. A method of determining the concentration or quantity of an analyte of interest in an analytical sample comprising:analysing a first set of samples that comprises a quality control sample and a plurality of analytical samples, wherein said quality control sample contains a plurality of calibrators at different known concentration levels and at least some of the analytical samples do not contain at least one of said calibrators, wherein said analysing obtains a signal for an analyte of interest in each of the analytical samples, and wherein said analysing obtains a signal for each known concentration level of said calibrators in said quality control sample;determining a calibration relationship based on said plurality of different known concentration levels of the calibrators and their respective ion signals obtained from analysing said quality control sample; anddetermining the concentration or quantity of the analyte of interest in at least one of said analytical samples using the signal detected for the analyte of interest in that analytical sample and the calibration relationship.

29. Automated apparatus having control circuitry and being configured to perform the method of any preceding claim.

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