Calibration of analyte amount in mass spectrometry

Abstract

A method of determining the concentration or quantity of an analyte of interest in an analytical sample comprising: analysing (e.g. mass analysing) a set of analytical samples that collectively contai

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority from and the benefit of United Kingdom patent application No. 2408095.4 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 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 set of analytical samples that collectively contain a plurality of different known concentration levels of at least one calibrator, wherein only a subset of said different known concentrations levels is present in each of the analytical samples, and wherein said mass analysing obtains, for each analytical sample, an ion signal for an analyte of interest and also an ion signal for each known concentration level of said at least one calibrator that is present in that analytical sample; determining a calibration relationship based on said plurality of different known concentration levels of the at least one calibrator and their respective ion signals obtained from mass analysing said set of analytical samples; 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. As the different known concentration levels of at least one calibrator are provided in the set of analytical samples, 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. 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 only a subset of said different known concentrations levels can be provided in each of the analytical samples and that the calibration relationship can be built up during the mass analysis of the set of analytical samples, whilst still providing an effective calibration relationship. Each of the analytical samples in the set of analytical samples contains a known concentration level that is unique to that analytical sample (within the set of analytical samples). The method may determine the calibration relationship based on all of said plurality of different known concentration levels of the at least one calibrator and their respective ion signals obtained from mass analysing said set of analytical samples. Each analytical sample may have an unknown quantity or concentration of the analyte of interest. Said set of analytical samples may be formed from consecutively mass analysed analytical samples. These 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. It is contemplated that one or more analytical sample that does not have said at least one calibrator may be mass analysed between analytical samples in said set of analytical samples. In such embodiments, it is preferred that the analytical samples forming said set of analytical samples are mass analysed within a set of n consecutively mass analysed analytical samples, where n is <20, <15, <10, <8, or <6. The number of analytical samples in each set of analytical samples may be >2; >3; >4; >5; or >6. The method may further comprise performing a cycle comprising: (a) mass analysing a further set of analytical samples that collectively contain a plurality of different known concentration levels of said at least one calibrator, wherein only a subset of said different known concentrations levels is present in each of these analytical samples, and wherein said mass analysing step obtains, for each of these analytical samples, an ion signal for the analyte of interest and also an ion signal for each known concentration level of said at least one calibrator that is present in that analytical sample; (b) updating said calibration relationship, or determining a new calibration relationship, using at least one of said plurality of different known concentration levels of the at least one calibrator in said further set of analytical samples and their respective ion signals obtained from mass analysing these analytical samples; 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 during said cycle. In other words, the updated calibration relationship if formed using the ion signals obtained prior to said cycle being performed and also the ion signals obtained 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 in said cycle. 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 method may comprise repeating said cycle a plurality of times, wherein each time the cycle is performed the calibration relationship from step (b) of the preceding cycle is updated. Each time the cycle is performed, step (b) may update the calibration relationship 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. 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 may be performed a further time in which a new calibration relationship is determined that does not use the ion signals 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. The method may comprise providing analytical samples in a batch of samples and repeating said cycle until all of the sets of analytical samples in the batch have been analysed. The batch of samples 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 method may comprise providing analytical samples in a plurality of batches of samples, and wherein either: (i) the set of analytical samples that are mass analysed first in each batch are used to form a new calibration relationship; or (ii) the set of analytical samples that are mass analysed first in at least some of the batches are used to update the calibration relationship obtained at the end of analysing the previous batch. Each time the cycle is performed, the updated or new calibration relationship may only 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. Alternatively, the updated or new calibration relationship could be used to determine the concentration or quantity of the analyte of interest in analytical samples that were mass analysed in other cycles. The number of analytical samples in the set of analytical samples that is mass analysed may be the same for all of the cycles that are performed. Alternatively, the number of analytical samples in the set of analytical samples that is mass analysed may be different in different cycles. 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 at least one calibrator includes a first concentration level above said range and / or a second concentration level below said range. Said different known concentration levels of the at least one calibrator may include at least one further concentration level between said first and second concentration levels. Each analytical sample in said set of analytical samples may only have a single one of said different concentration levels therein. Alternatively, all of the analytical samples in said set of analytical samples may include at least one of said different concentration levels that is the same. For example, all of the analytical samples in said set of analytical samples may include said first and / or second concentration level. Said at least one of the different concentration levels that is the same may be used as an internal standard. The at least one calibrator may be at least one compound that produces an ion signal 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 at least one 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 calibrator compounds are referred to herein as stable isotope labelled calibrator (SILC) compounds. However, other compounds may be used as the at least one calibrator instead, such as a compound that is different to the analyte of interest but is in the same family of compounds. Said at least one calibrator may be a single calibrator, wherein all of the analytical samples in said set of analytical samples have different known concentrations of said single calibrator. In these embodiments the calibrator in each of the analytical samples has the same molecular mass. The method may comprise mass analysing the analytical samples in the set of analytical samples sequentially and in a manner such that sequentially mass analysed analytical samples have progressively higher or progressively lower concentrations levels of said calibrator in them. Alternatively, the method may comprise mass analysing the analytical samples in the set of analytical samples sequentially, wherein the analytical samples having the different concentrations levels are mass analysed in a random order. In embodiments in which said cycle is performed, each time the cycle is performed the analytical samples in the set of analytical samples may be mass analysed in the same order of known concentrations as said random order. Alternatively, each time the cycle is performed the analytical samples in the set of analytical samples may be mass analysed in a new random order. Said at least one calibrator may be a plurality of calibrators, wherein all of the analytical samples in said set of analytical samples have different known concentrations of said calibrators. In these embodiments the plurality of calibrators have different molecular masses. Each of the analytical samples in the set of analytical samples may include multiple ones of said calibrators having different respective known concentrations. At least one of the calibrators may be present at the same known concentration in all of the analytical samples in the set of analytical samples, and one or more further calibrator may be provided in at least some of the samples in the set of analytical samples with concentrations that are different for different ones of these samples. The one of more calibrators described herein may be applied to all analytical samples, or to every nth analytical sample where n is an integer, or to random analytical samples. The mass spectrometer may be operated to always mass analyse the one of more calibrators if they are present, or it may be operated to not mass analyse the one or more calibrators in some of the analytical samples. The analytical samples may be mass analysed using liquid chromatography mass spectrometry; optionally wherein each of the analytical samples in said set of analytical samples is mass analysed using tandem mass spectrometry by monitoring an MRM transition for said analyte of interest and an MRM transition for said at least one calibrator. 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. The method may be performed by automated machinery that: (i) receives analytical samples; (ii) adds said plurality of different known concentration levels of the at least one calibrator to a plurality 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 as described herein. 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 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 samples are mass analysed, it is contemplated that the samples may be analysed using other techniques. 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: analysing a set of analytical samples that collectively contain a plurality of different known concentration levels of at least one calibrator, wherein only a subset of said different known concentrations levels is present in each of the analytical samples, and wherein said analysing obtains, for each analytical sample, a signal for an analyte of interest and also a signal for each known concentration level of said at least one calibrator that is present in that analytical sample; determining a calibration relationship based on said plurality of different known concentration levels of the at least one calibrator and their respective signals obtained from analysing said set of analytical samples; 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 second 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 second 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 calibrator(s). Accordingly, said analysing may comprise ion mobility analysing the set of analytical samples, wherein said ion mobility analysing obtains, for each analytical sample, an ion signal for the analyte of interest and also an ion signal for each known concentration level of said at least one calibrator that is present in that analytical sample. The calibration relationship may be determined based on said plurality of different known concentration levels of the at least one calibrator and their respective ion signals obtained from ion mobility analysing said set of analytical samples. The step of determining the concentration or quantity of the analyte of interest in at least one of said analytical samples may use 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. The present invention therefore 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; Figs. 4 and 5A-5E illustrate the concentrations of calibrator and analyte in different samples according to embodiments of the present invention; Figs. 6A-6D show example ion-chromatograms for an analyte of interest and different calibrators; Fig. 7 shows a calibration relationship calculated based on the peak areas measured from the ion chromatograms shown in Figs. 6B-6D; Fig. 8 shows a 96-well sample plate according to another embodiment of the present invention; Figs. 9 and 10 illustrate the concentrations of calibrator and analyte in different samples according to embodiments of the present invention; and Fig. 11 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 prepared in matrix 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, different known concentrations of a calibrator compound are provided in different, respective 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 illustrated example, six different known concentrations of the calibrator compound are used (shown as SILC Levels 1-6), although it will be appreciated that fewer or a greater number of different known concentrations may be used in a corresponding fewer or a greater number of analytical samples. Each set of six consecutive analytical samples on the sample plate is provided with the six different known concentrations of the calibrator compound, wherein each of these consecutive analytical samples is provided with a unique concentration of the calibrator compound. The term consecutive analytical samples means samples that are analysed consecutively. In this example the samples in the sample wells are analysed in an order from left to right for each row, starting with row A and then proceeding downwards to row H. However, the six consecutive analytical samples in each set are not necessarily arranged in six consecutive sample wells on the sample plate, as one or more other non-analytical sample may be arranged in one or more respective sample well so that it is analysed in between analysing different analytical samples in a set of six consecutive analytical samples. For example, a quality control sample may be provided in a sample well such that it is analysed between analysing different analytical samples in a set of six consecutive analytical samples. The calibrator compound should ideally behave, chemically, in as similar manner as possible to the analyte of interest. As such, the calibrator compound 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 compound will be referred to herein as a stable isotope labelled calibrator (SILC) compound. In the following embodiments the calibrator compound will be described as a SILC compound, although it will be appreciated that other types of calibrator compound may be used instead, such as a compound that is different to the analyte of interest but in the same family of compounds. 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. A set of six analytical samples having six different concentrations of the SILC compound is provided in the six consecutive sample wells from A7-A12. Each of the subsequent sets of six consecutive analytical samples on the sample plate are also provided with the six different concentrations of the SILC compound. More specifically, a set of six analytical samples having six different concentrations of the SILC compound are provided in each of the following sets of sample wells: B1-B6; B7-B12; C1-C6; C8-D1; D2-D7; D8-E1; E2 and E4-E8; E9-F2; F3-F8; F9-F11 and G1-G3; and G4-G9, G10-H3. 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. A set of five analytical samples having five different concentrations of the SILC compound are provided in the remaining five consecutive sample wells from H4-H8. An internal standard may also be added, at the same concentration, to each of the analytical samples in the batch. 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. The internal standard compound and the calibrator compound may be different, at least in the sense that they are distinguishable by the mass spectrometer. For example, the SILIS compound and the SILC compound may be the same chemical compound, but wherein one or more atoms in the chemical structure of the SILC compound is a different isotope to in the SILIS compound. 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 analytical samples from sample wells A7-A12, wherein for each of the analytical samples the mass spectrometer monitors a different MRM transition in order to detect an ion signal for each of: the analyte of interest, the SILC compound, and optionally the SILIS compound if one has been added to the analytical samples. 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 the SILC compound, 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 compound 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 compound in the sample. A different MRM transition for the SILC compound 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 compound 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 compound that is being detected, since it is highly unlikely that a precursor ion other than the SILC compound would give rise to ion signals for both MRM transitions. For the SILIS compound, 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 compound 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 compound in the sample. A different MRM transition for the SILIS compound 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 compound 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 compound that is being detected, since it is highly unlikely that a precursor ion other than the SILIS compound would give rise to ion signals for both MRM transitions. Preferably, the MRM transition(s) for each of the analyte of interest, the SILIS compound, and the SILC compound are selected such that ions will be detected at the detector if these undergo the same manner of fragmentation, i.e. these compounds fragment in the same place(s) of the chemical structure. The MRM transitions(s) for the analyte of interest, the SILIS compound, and the SILC compound 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 analytical 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 compound, and the SILC compound. The mass spectrometer determines the abundance of ions in the ion peak for each ion chromatograms, 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 compound, and the SILC compound and as such a relative response factor may be calculated for each of the analyte of interest and the SILC compound (relative to the SILIS compound). For example, the abundance of the analyte of interest may be divided by the abundance of the SILIS compound so as to obtain a relative response factor for the analyte of interest, and similarly the abundance of the SILC compound may be divided by the abundance of the SILIS compound so as to obtain a relative response factor for the SILC compound. The relative response factor for the SILC compound is then used to calculate an apparent concentration for the SILC compound, e.g. by multiplying the known concentration of the SILC compound in that analytical sample by the relative response factor determined for the SILC compound. 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 compound that gave rise to this signal. This process is performed on each set of the six analytical samples having the six different known concentrations of the SILC compound therein, so as to obtain a data pair for each of the six concentrations of the SILC compound. 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 signals, 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 of the six analytical samples (and optionally the preceding 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. Referring back to Fig. 3, the method then proceeds to sequentially analyse the remaining samples in the other sample wells of the sample plate. For example, the method will next sequentially analyse the samples from sample wells B1-B6 in a corresponding manner to that described above. The mass spectrometer will therefore determine a data pair consisting of the ion signal detected (ion abundance) and the apparent concentration or quantity of the SILC compound that gave rise to this signal for each of these analytical samples. Each of these data pairs may be used to update the previously calculated calibration relationship, e.g. by inclusion of each subsequent data pair in the least squares fitting process described above. The calibration relationship may be updated each time a new data pair from a newly analysed analytical sample is obtained, or only after a predetermined number of such data pairs have been obtained. For example, each time a complete set of the six consecutive analytical samples has been analysed, the resulting data pairs may be used to update the calibration relationship and then that updated calibration relationship may be used to determine the quantity or concentration of the analyte of interest in each of the analytical samples in the set of analytical samples used to update the calibration relationship. The calibration relationship may be updated by recalculating the calibration relationship using only a preselected number of the most recently obtained data pairs for each of the known concentrations of the SILC compounds. For example, the calibration relationship may be recalculated using only the last five data pairs obtained for each of the known concentrations of the SILC compounds. Fig. 4 illustrates the known concentrations of the SILC compound in 12 consecutive analytical samples relative to the expected range of unknown concentrations of the analyte of interest in the analytical samples. This example illustrates a first set of six consecutive analytical samples having the six different known concentrations of the SILC compound (i.e. SILC levels 1-6), and a second set of six consecutive analytical samples having the six different known concentrations of the SILC compound. 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 SILC compound 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 SILC compound relatively evenly spaced therebetween. However, it will be appreciated that the known concentrations of the SILC compound 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 SILC compound 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 analyte 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. In the embodiment described above, the consecutive analytical samples in each set of consecutive analytical samples have been described as being provided with progressively increasing known concentrations of the SILC compound, e.g. as shown in Fig. 4. However, it is contemplated that the consecutive analytical samples in each set of consecutive analytical samples may be provided with known concentrations of the SILC compound that progressively decrease, e.g. as shown in Fig. 5A, or that vary in another manner such as is shown in Figs. 5B-5D. Fig. 5B shows an embodiment in which the known concentrations of the SILC compound vary in a random pattern across the analytical samples in each set of consecutive analytical samples, but wherein that pattern is repeated for each set of consecutive analytical samples. Fig. 5C shows an embodiment in which the six known concentrations of the SILC compound are provided in each set of consecutive analytical samples, but wherein they are provided in a random order across each set (i.e. not in a repeating pattern). Fig. 5D shows an embodiment in which the six known concentrations of the SILC compound are provided in a random order across all of the analytical samples (i.e. each set of six analytical samples may not include all six different concentrations). These embodiments, in which the order of the concentrations is pseudo-random, enable the detection of temporal or systematic changes that affect the signals measured by the mass spectrometer. For example, automated machinery having multiple pipettes may be used for extracting and transferring samples from the sample wells, and if samples having any given one of the concentrations of the SILC compound are always sampled by the same pipette then it would not be possible to detect if that pipette has become defective. In contrast, if the order of the concentrations is pseudo-random, as described above, then if one of the pipettes becomes defective then the presence of a defect could be detected in the calibration data. As mentioned above, although a set of six consecutive analytical samples have been described as having six different concentrations of the SILC compound, the number of consecutive analytical samples having different concentrations of the SILC compound may be different to this. Although the SILC compound has been described as being present in every analytical sample, this need not be the case and therefore the different known concentrations of the SILC compound need not be in analytical samples that are consecutively analysed. Although embodiments have been described that use different known concentrations of a SILC compound having a single molecular mass in each set of analytical samples, it is contemplated that SILCs having different molecular masses and different respective known concentrations may be used in the different analytical samples in each set of analytical samples. For example, Fig. 5E illustrates an embodiment corresponding to that in Fig. 4, except that the SILCs having different concentrations also have different molecular masses. The SILCs having different molecular masses may be the same compound but in which one or more atoms in the chemical structure have different isotopes. For example, the first known concentration of the SILC compound in the set of analytical samples may have a first molecular mass, the second known concentration of the SILC compound in the set may have a second different molecular mass, the third known concentration of the SILC compound in the set may have a third different molecular mass, and so on. It will be appreciated that slightly different MRM transitions will be required to mass analyse each of these different molecular masses. An example of this is described immediately below. 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 analytical samples in the set of consecutive analytical samples having different concentrations of the SILC compound. In this example, the SILC compound has a different molecular mass in the three analytical samples, by adding one of di-, tri-, and penta-deuterated testosterone to a respective one of the three analytical samples. 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 a first analytical sample, in the set of three analytical samples, is analysed the mass spectrometer will monitor the MRM transition for the analyte of interest and also the MRM transition for di-deuterated testosterone, and produce ion chromatograms for these MRM transitions. Similarly, when a second analytical sample, in the set of three analytical samples, is analysed the mass spectrometer will monitor the MRM transition for the analyte of interest and also the MRM transition for tri-deuterated testosterone, and produce ion chromatograms for these MRM transitions. Similarly, when a third analytical sample, in the set of three analytical samples, is analysed the mass spectrometer will monitor the MRM transition for the analyte of interest and also the MRM transition for penta-deuterated testosterone, and produce ion chromatograms for these MRM transitions. Figs. 6A-6D show example ion-chromatograms for the above described MRM transitions. More specifically, Fig. 6A 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. 6B 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. 6C 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. 6D shows an ion-chromatogram for the analysis of 10.0 ng / mL pentadeuterated 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. 7 shows a calibration relationship that is calculated based on the peak areas measured from the ion chromatograms shown in Figs. 6B-6D 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. 6A was determined and, using the calibration relationship shown in Fig. 7, it was determined that the analyte of interest has a concentration of 2.85 ng / mL. Fig. 8 shows a 96-well sample plate according to another embodiment of the present invention. This embodiment is similar to that shown in Fig. 3 in that the analytical samples in each of the sets of consecutive analytical samples have a unique concentration of a SILC compound therein. However, in this embodiment all of the analytical samples also contain a first SILC at the same first concentration and a second SILC at the same second concentration. It will be appreciated that the SILCs in any given analytical sample must be distinguishable from each other by different MRM transitions being monitored by the mass spectrometer, e.g. by having different molecular masses. In this embodiment a SILIS compound may or may not be added to any of the analytical samples. In the example shown in Fig. 8, 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. A set of four consecutive analytical samples having different combinations of SILCs is provided in the four consecutive sample wells from A7-A10. More specifically, SILCs having six different concentrations are present in the analytical samples in sample wells A7-A10, where each analytical sample in the set of four samples has a unique combination of the six different concentrations of SILCs. These six different concentrations will be referred to as SILC1 to SILC6 for convenience. In the example shown, the analytical sample in sample well A7 contains SILC1, SILC2 and SILC6. The analytical sample in sample well A8 contains SILC1, SILC3 and SILC6. The analytical sample in sample well A9 contains SILC1, SILC4 and SILC6. The analytical sample in sample well A10 contains SILC1, SILC5 and SILC6. In this example, the higher the SILC number is the higher the concentration is (i.e. SILC2 has a higher concentration than SILC1 etc.), although that need not be the case. Those skilled in the art will recognise that SILC1 and SILC6 must be distinguishable from each other by the mass spectrometer, and also each distinguishable from each of SILCs2-5. As such, SILC1 and SILC6 have molecular masses that are different to each other and that are also different to the molecular masses of each of SILCs2-5. However, SILCS 2-5 need not necessarily be distinguishable from each other by the mass spectrometer as they are not present in the same analytical sample. Accordingly, SILCs2-5 may all have the same molecular mass, or at least some of SILCs2-5 may have different molecular masses. Further sets of four consecutive analytical samples that have the different combinations of SILCs were provided on the sample plate. For example, a set of four analytical samples having four unique combinations of SILCs is provided in each of the following sets of sample wells: A11-B2; B3-B6; B7-B10; B11-C2; C3-C6; C8-C11; C12-D3; D4-D7; D8-D11; D12-E2 and E4; E5-E8; E9-E12; F1-F4; F5-F8; F—F11 and G1; G2-G5; G6-G9; G10-H1; and H2-H5. 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 blank sample is provided at sample well H9. A partial set of three analytical samples having different combinations of SILCs is provided in the remaining three consecutive sample wells from H6-H8. In use the samples on the sample plate are analysed as has been described above in relation to Fig. 3, except that because any given analytical sample in the embodiment shown in Fig. 8 has three SILCs the mass spectrometer is required to monitor three respective MRM transitions for those three SILCs. After the first set of four analytical samples had been 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, e.g. the quality control samples, may then be calibrated using this relationship. In a corresponding manner to that previously discussed, after the analysis of each set of four consecutive analytical samples (or after the analysis of each of those analytical samples), the calibration relationship is updated using the data-pairs obtained from the analysis of SILCs1-6. The updated calibration relationship is then used to calculate the concentration or quantity of the analyte of interest in each of the analytical samples in the set of samples that was used to update the calibration relationship. The analytical samples in sample wells H6-H8 form an incomplete one of the sets of analytical samples. The updated calibration relationship obtained from the analysis of the immediately preceding set of analytical samples may be used to determine the concentrations of the analytes of interest from sample wells H6-H8. Fig. 9 illustrates the known concentrations of the SILCs in 12 consecutive analytical samples (i.e. from three of the sets of analytical samples) relative to the expected range of unknown concentrations of the analyte of interest in the analytical samples. This example illustrates a first set of four consecutive analytical samples having the six different known concentrations of the SILCs, a second set of four consecutive analytical samples having the six different known concentrations of the SILCs, and a third set of four consecutive analytical samples having the six different known concentrations of the SILCs. The vertically elongated rectangles represents the range of concentrations of the analyte of interest that might be expected in the analytical samples. It can be seen that for any given one of the sets of analytical samples, 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. In this embodiment the highest and lowest concentrations of the SILCs (i.e. SILC1 and SILC6) are provided in each analytical sample. However, this need not be the case, e.g. as is shown by Fig. 10. 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. 11. Fig. 11 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 SILC(s) 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 SILC compounds, it will be appreciated that the SILC compound 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 an MRM mode. However, other mass analysis techniques and / or mass spectrometer may be used to obtain ion peaks for the analyte of interest and calibrator(s) 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, 5 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 proteins. 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 10 corresponding manner to that described. For example, each analyte of interest may have its own respective calibrator(s). 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 15 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 set of analytical samples that collectively contain a plurality of different known concentration levels of at least one calibrator, wherein only a subset of said different known concentrations levels is present in each of the analytical samples, and wherein said mass analysing obtains, for each analytical sample, an ion signal for an analyte of interest and also an ion signal for each known concentration level of said at least one calibrator that is present in that analytical sample;determining a calibration relationship based on said plurality of different known concentration levels of the at least one calibrator and their respective ion signals obtained from mass analysing said set of analytical samples; 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.

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, further comprising performing a cycle comprising:(a) mass analysing a further set of analytical samples that collectively contain a plurality of different known concentration levels of said at least one calibrator, wherein only a subset of said different known concentrations levels is present in each of these analytical samples, and wherein said mass analysing step obtains, for each of these analytical samples, an ion signal for the analyte of interest and also an ion signal for each known concentration level of said at least one calibrator that is present in that analytical sample;(b) updating said calibration relationship, or determining a new calibration relationship, using at least one of said plurality of different known concentration levels of the at least one calibrator in said further set of analytical samples and their respective ion signals obtained from mass analysing these analytical samples; 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.

4. The method of claim 3, 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).

5. The method of claim 3 or 4, comprising repeating said cycle a plurality of times, wherein each time the cycle is performed the calibration relationship from step (b) of the preceding cycle is updated.

6. The method of claim 3, 4 or 5, wherein each time the cycle is performed, step (b) updates the calibration relationship using only a preselected number of most recently obtained ion signals for each of the different concentration levels.

7. The method of any one of claims 3-6, wherein the cycle is repeated for a preselected 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 obtained in any preceding cycles.

8. The method of any one of claims 3-6, wherein the cycle is only repeated for up to a pre-selected number of cycles or for a pre-selected duration of time.

9. The method of any one of claims 3-6, comprising providing analytical samples in a batch of samples and repeating said cycle until all of the sets of analytical samples in the batch have been analysed.

10. The method of claim 9, comprising providing analytical samples in a plurality of batches of samples, and wherein either:(i) the set of analytical samples that are mass analysed first in each batch are used to form a new calibration relationship; or(ii) the set of analytical samples that are mass analysed first in at least some of the batches are used to update the calibration relationship obtained at the end of analysing the previous batch.

11. The method of any one of claims 3-10, wherein each time the cycle is performed, the updated or new calibration relationship is only 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.

12. 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 at least one calibrator includes a first concentration level above said range and / or a second concentration level below said range.

13. The method of claim 12, wherein said different known concentration levels of the at least one calibrator includes at least one further concentration level between said first and second concentration levels.

14. The method of any preceding claim, wherein each analytical sample in said set of analytical samples only has a single one of said different concentration levels therein.

15. The method of any one of claims 1-13, wherein all of the analytical samples in said set of analytical samples include at least one of said different concentration levels that is the same.

16. The method of any preceding claim, wherein each of said at least one calibrator 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.

17. The method of any preceding claim, wherein said at least one calibrator is only a single calibrator, and wherein all of the analytical samples in said set of analytical samples have different known concentrations of said single calibrator.

18. The method of claim 17, comprising mass analysing the analytical samples in the set of analytical samples sequentially and in a manner such that sequentially mass analysed analytical samples have progressively higher or progressively lower concentrations levels of said calibrator in them.

19. The method of claim 17, comprising mass analysing the analytical samples in the set of analytical samples sequentially, wherein the analytical samples having the different concentrations levels are mass analysed in a random order.

20. The method of any one of claims 1-16, wherein said at least one calibrator is a plurality of calibrators, and wherein all of the analytical samples in said set of analytical samples have different known concentrations of said calibrators.

21. The method of claim 20, wherein each of the analytical samples in the set of analytical samples includes multiple ones of said calibrators having different respective known concentrations.

22. The method of claim 21, wherein at least one of the calibrators is present at the same known concentration in all of the analytical samples in the set of analytical samples; and one or more further calibrator is provided in at least some of the samples in the set of analytical samples with concentrations that are different for different ones of these samples.

23. 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 in said set of analytical samples is mass analysed using tandem mass spectrometry by monitoring an MRM transition for said analyte of interest and an MRM transition for said at least one calibrator.

24. The method of any preceding claim, wherein the method is performed by automated machinery that:(i) receives analytical samples;(ii) adds said plurality of different known concentration levels of the at least one calibrator to a plurality 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.

25. The method of claim 24, 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.

26. The method of any preceding claim, wherein each analytical sample has an unknown quantity or concentration of the analyte of interest.

27. A method of determining the concentration or quantity of an analyte of interest in an analytical sample comprising:analysing a set of analytical samples that collectively contain a plurality of different known concentration levels of at least one calibrator, wherein only a subset of said different known concentrations levels is present in each of the analytical samples, and wherein said analysing obtains, for each analytical sample, a signal for an analyte of interest and also a signal for each known concentration level of said at least one calibrator that is present in that analytical sample;determining a calibration relationship based on said plurality of different known concentration levels of the at least one calibrator and their respective signals obtained from analysing said set of analytical samples; 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.

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

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