WO2024097357A1 - A high-throughput low-density lipoprotein cholesterol level screening method - Google Patents

Abstract

Described herein is a method of extracting and quantitating low density lipoprotein cholesterol (LDL-C) in a blood sample. The sample is first diluted or rehydrated in a reagent 1 to provide a diluted sample. The diluted sample is then mixed with an aqueous reagent 2 including an LDL-C protecting agent, a cholesterol esterase enzyme, a cholesterol oxidase enzyme, and optionally a peroxidase for a time and temperature to degrade non-LDL cholesterol while maintaining LDL-C to provide a reacted sample comprising the LDL-C. The LDL-C is then extracted from the reacted sample by mixing a volume of the reacted sample with a reagent 3 including a water-immiscible organic liquid an internal standard and removing the water-immiscible organic liquid phase sample including the LDL-C and the internal standard (IS). The cholesterol and esters are separated and area of areas of sample LDL-free cholesterol, sample LDL cholesterol esters, free cholesterol internal standard and cholesterol esters internal standard are determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Finally, total LDL-C is quantitated.

Description

A HIGH-THROUGHPUT LOW-DENSITY LIPOPROTEIN CHOLESTEROL LEVEL SCREENING METHOD CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application 63/422,471 filed on November 4, 2022, which is incorporated herein by reference in its entirety. BACKGROUND [0001] Coronary artery disease (CAD) is the leading annual cause of death in the United States and the world at large, making the prevention and treatment of CAD a public health mission. Elevated concentrations of low-density lipoprotein cholesterol (LDL-C) in circulating blood serum is the most widely accepted biological measurement that indicates an individual has a significant risk of developing CAD in their lifetime. High levels of LDL-C in the blood cause cholesterol precipitation onto arterial walls, where continual long-term build up leads to the development of plaques that eventually obstruct blood flow and directly cause life-threatening cardiovascular events. Well-known risk factors for high LDL-C levels include smoking, lack of exercise, poor diet, obesity, diabetes and high-blood pressure. [0002] Medical clinics typically calculate LDL-C levels using the Friedwald equation that requires inputting total cholesterol, high-density lipoprotein cholesterol (HDL-C) and triglyceride concentrations. These values can be measured at local medical laboratories. In the past two decades, homogenous methods have also been introduced that directly measure LDL-C concentration in a single assay. All of these methodologies rely on a well-established set of enzymes and chemicals to produce a colored liquid biproduct from the target analyte after interfering substances or cholesterol arising from non-targeted populations of macromolecules that are destroyed or masked. The absorbances of the resulting solutions are determined and compared to a calibration curve to directly calculate the sought-after analyte concentration values. Lipid panels measuring LDL-C are routinely ordered for middle-age or at-risk adults, as visits to medical providers become more regular to monitor health and prevent disease. [0003] A common autosomal co-dominant genetic disorder, named familial hypercholesterolemia (FH), is a leading cause of LDL-C metabolism dysfunction that leads to elevated blood LDL-C levels. With an incidence of approximately 1 in 250, individuals afflicted with FH manifest increased LDL-C blood levels from birth, and gradually develop cholesterol precipitation onto arterial walls and arterial plaque buildup. This accordingly leads to a substantial increased risk of a life-threatening cardiovascular disorder later during adulthood. Statin-based pharmaceuticals offer a straightforward and well-tolerated medical intervention that can reduced LDL-C levels by as much as 50% in order to prevent the development of CAD. The most effective preventative treatment plan would start in childhood between 9-11 years old, however widescale screening and detection of FH is not currently performed in the US. [0004] What is needed are improved methods of determining LDL-C levels that are particularly useful for high throughput FH screening. SUMMARY [0005] In an aspect, a method of extracting and quantitating low density lipoprotein cholesterol (LDL-C) in a blood sample comprises: diluting or rehydrating the blood sample in an aqueous reagent 1 (R1) to provide a diluted sample having a first dilution factor (DF1); incubating a volume (SV1) of the diluted sample with an aqueous reagent 2 (R2) to provide a reaction solution having a second dilution factor (DF2), wherein incubating is for a time and temperature to degrade non-LDL cholesterol while maintaining LDL-C to provide a reacted sample comprising the LDL-C, wherein R2 comprises an LDL-C protecting agent, a cholesterol esterase enzyme, a cholesterol oxidase enzyme, and optionally a peroxidase; optionally arresting the reaction in the reacted sample to inhibit further cholesterol oxidase enzyme activity; extracting the LDL-C from the reacted sample by mixing a volume of the reacted sample (SV2) and a reagent 3 (R3) and removing a water-immiscible organic liquid phase sample comprising the LDL-C and an internal standard (IS), wherein R3 comprises the water-immiscible organic liquid, an organic liquid miscible with both water and the water-immiscible organic liquid, and the IS, wherein the IS comprises isotope-labeled cholesterol ester (IS1) and isotope- labeled free cholesterol (IS2), and wherein the water-immiscible organic liquid phase sample has a third dilution factor (DF3); optionally drying a portion of the water-immiscible organic liquid phase sample and reconstituting it in a mobile phase to provide a chromatography sample; separating cholesterol esters (CE) and free cholesterol (FC) in the water-immiscible organic liquid phase sample or the chromatography sample and determining areas of sample LDL-free cholesterol (A FC-LDL), sample LDL cholesterol esters (A CE-LDL), free cholesterol internal standard (A _FC-IS) and cholesterol esters internal standard (A _CE-IS) by liquid chromatography-tandem mass spectrometry (LC-MS/MS); and quantitating total LDL-C as the sum of A FC-LDL and A CE-LDL in the sample normalized by A _FC-IS and A _CE-IS. BRIEF DESCRIPTION OF THE DRAWINGS [0006] Fig. 1 shows measured LDL-C values compared to expected values. The dotted line is the calculated linear curve fit possessing a correlation coefficient of 0.983 and a slope of 0.979 ± 0.026. [0007] Fig. 2 shows a least squares linear regression model. Measured LDL-C values calculated without correction for the recovery factor were plotted against serum values for corresponding samples. The slope of 0.6913 is equivalent to the fractional percent recovery factor, PR. [0008] Fig. 3 shows measured LDL-C values in dried blood spots compared to corresponding serum values. The dotted line is the calculated linear curve fit possessing a correlation coefficient of 0.958 and a slope of 0.999 ± 0.053. [0009] The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims. DETAILED DESCRIPTION [0010] Described herein is a process of determining LDL-C levels which can be used for FH screening. The method is straight-forward and reproducible. Advantageously, the method is adapted for high throughput screening in a 96-well plate setting, for example. In an aspect, a small amount of blood is collected on filter papers cards, similar to well-established newborn screening methods for congenital disorders. Such sample cards are stable at room-temperature after a short drying period at ambient conditions. These sample cards are then sent to a medical laboratory for analysis by routine mail. Although this method of sample collection can be performed at a medical facility, such as a hospital or clinic, it can also be done at home or at schools by non-professionals to make screening easily accessible for the testing of children. Described herein is a method to rehydrate and release the blood products from these dried blood spots such that subsequent LDL-C measurements are closely proportional to those in the original sample. In another aspect, traditional venous blood samples may be obtained at a medical facility, for example for the purpose of forming serum or plasma samples, to measure LDL-C by the disclosed method, which thereby averts the need for rehydration and release steps. Collection of blood products in liquid form, however, generally requires storing samples at 4°C or below. [0011] For both sample collection procedures, liquid chromatography (LC) – tandem mass spectrometry (MS) is employed to detect and quantify the LDL-C levels in the original or rehydrated blood samples, taking advantage of this technique’s sensitivity to assay the small volume of collected sample. Because LDL-C is not only used as a biomarker for FH, but also for CAD in general, this process can additionally be used to perform an initial evaluation of CAD risk in adults and to monitor patients of any age that are being treated for high cholesterol. [0012] Described herein is a process to measure LDL-C concentrations in a collected blood sample, specifically serum or plasma, or in the form of a dried blood spot. Cholesterol found in the low-density lipoproteins is indistinguishable from that contained in other lipoprotein particles, such as high-density lipoprotein that typically constitutes 20-40% of the total cholesterol. As such, sample processing steps serve to both isolate LDL-C from other lipoprotein cholesterols and to dilute the LDL-C to measurable range. LDL-C levels in such processed samples are measured by LC – tandem MS, and the values in the original samples are calculated by the provided equations. Cholesterol contained in low-density lipoprotein exists molecularly both as free cholesterol and a heterogenous mixture of cholesterol ester species, therefore both free cholesterol and cholesterol esters are measured in a single analysis. SAMPLE COLLECTION [0013] In the first sampling method, blood is obtained by a venous draw such as at a licensed medical facility into a commercially available, standardized collection tube, for example, a tube that isolates serum or plasma from the drawn blood, utilized per manufacturer instructions. A minimum blood collection volume of approximately 0.1 mL is typically used, although much larger volumes may be obtained according to blood collection tube instructions. 0107668.0116 P220319WO01 These samples may be stored at 4°C for up to a week, or, if in serum or plasma form, at -20°C up to many months before LDL-C analysis. As used herein, a venous blood sample is a liquid blood sample, such as whole blood, serum or plasma, and is distinguished from a dried blood spot. [0014] In a second blood collection method, a dried blood spot is created. A sterile lancet may be utilized to pierce the skin of the test recipient, typically on a fingertip, where a blood droplet is allowed to briefly collect on the patient’s skin before being spotted on Grade 903 WhatmanTM filter paper or equivalent. Saturation of the filter paper is required, where an approximate 60 µL of blood formed circle of spot is visible on each side of the collection paper. To complete the dried blood spot creation process, the collected sample is dried at room temperature for a minimum of three hours. Dried blood spot samples are taken or sent to a laboratory at room temperature. Samples may be stored up to 2-4 weeks at room temperature, up to 2-3 months at 4°C or up to 2 years at -20°C or -80°C before LDL-C analysis. Such dried blood spot sample collection may be performed at any location, for example at a school or at the recipient’s residence, by a non-professional, including by the test recipient themselves or their legal guardians. PROCESSING STEP 1: PREPARING A DILUTED BLOOD SAMPLE SOLUTION [0015] In an aspect, for a venous blood sample, processing step 1 comprises dilution of the blood sample into an aqueous reagent, referred to as reagent 1 (R1). The sample dilution occurring during this processing step, referred to as dilution factor 1 (DF1), is the quotient of the final volume divided by the utilized original sample volume (SVo). The original sample volume is the volume of serum or plasma, as opposed to volume of whole blood, used in processing step 1. ^^^^^ ^^^^^^^ ^^ ^^^^^^^^ 1 (μ^) ^^^^^^^^ ^^^^^^ 1 =

[0016] For dried blood spot samples, processing step 1 comprises rehydration of the dried blood product along with its simultaneous release and dilution into R1. A circular punch is removed from the saturated portion of the blood spot and deposited into a multiwell plate, typically using either a hand puncher device or an automated puncher machinery that is sold commercially. Circular punches range in diameter from about 1/32th to 1/4^th inch and contain a known amount of the original blood product volume. A typical punch diameter taken from dried blood spots is 1/8^th inch that contains 3.2 μL of whole blood. Whole blood is comprised of 45 – 60% serum or plasma. Using an average population value of 50% serum in a given whole blood sample, a 1/8^th inch dried blood spot punch, constituted of 3.2 μL of whole blood, thereby contains 1.6 μL of serum or plasma that is the original sample volume (SVo), used in the equations below. [0017] Once all samples to be examined are punched, lipoprotein release and dilution to homogeneity occurs as describe above for plasma and serum samples, except that dried blood spot punches may be forcibly immersed in the R1 solution before agitation by centrifuging the sample plate for a minimum at 1 minute at 250 x g or greater. This process also results in a dilution factor 1 (DF1) that is calculated in an identical manner as above. [0018] For high-throughput processing, sample dilutions may occur in a multiwell plate (e.g., a 96 well plate) capable of holding volumes from 0.05 mL to 0.30 mL, using calibrated multi-channel pipettes or automated liquid handling systems (e.g., Perkin Elmer Zephyr® G3 automated workstation or Apricot Designs Personal PipettorTM Workstation) for liquid transfers. Mixing to homogeneity and equilibration typically complete the processing step 1. The plate may be covered with a commercially available seal or a plastic cap-mat and shaken for 15 minutes to 4 hours, at temperatures from 4°C to 40°C, at a rotation speed from 250 rotations per minute (rpm) to 2,000 rpm. [0019] R1 is an aqueous reagent which may comprise several components. The first component is a buffer capable of maintaining a solution pH at approximately 6.0 to 8.0, but set to an ideal pH of approximately 7.0-7.5, that is reconstituted to a final concentration from 5 – 200 mM buffer. Exemplary buffers include Tris, Bis-Tris, PIPES, HEPES, and sodium phosphate buffer salts. The second component is a non-buffered ionic salt reconstituted to a final concentration from 10 mM – 400 mM. Examples include NaCl, KCl, MgCl2, Mg2SO4, and combinations thereof. A third component comprises a reducing agent reconstituted at a concentration of 0.5 mM – 50 mM. Examples include (tris(2-carboxyethyl)phosphine) TCEP, dithiothreitol (DTT), and β-mercaptoethanol. Additional agents may be added that do not alter the stability or release of the blood product lipoproteins and may include sugar concentrations from 1% to 10% such as sucrose or glycerol, or mild detergents such as sodium deoxycholate at concentrations below 0.1% (w/v). Exemplary R1 reagents are available commercially through chemical supply companies. PROCESSING STEP 2 [0020] Sample processing step 2 comprises further dilution and incubation of the solution that results from sample processing step 1. A volume of the diluted sample, abbreviated SV1, ranging from 1 μL to 10 μL, is added to aqueous reagent 2 (R2). Volumes of R2 may be in the range of 50 μL to 300 μL. The resulting solution is maintained at 0°C to 10°C, so as long as the enzymes in R2 are non-reactive towards the samples until incubation at an elevated temperature is initiated. [0021] The additional sample dilution occurring during processing step 2, hereby termed dilution factor 2 (DF2), is the quotient of the final volume divided by the added volume. ^^^^^ ^^^^^^ ^^ ^^^^^^^^ 2 (μ^) ^^^^^^^^ ^^^^^^ 2 (^^2) = ^^^^^^ ^^ ^^^^^^^ ^^^^^^ ^^^^ ^^^^^^^^^^ ^^^^ 1 (^^1) ( μ^)

[0022] Reagent 2 (R2) comprises several components that are formulated specifically to allow the reaction of non-LDL cholesterol with cholesterol-destroying enzymes, which thereby leaves only LDL cholesterol intact and available for measurement. [0023] In an aspect, R2 comprises an LDL-C protecting agent which deprotects non-LDL cholesterol while it protects LDL-C. In an aspect, R2 comprises a mixture of surfactants at a final concentration of 0.05 – 1.0% (w/v). A combination of polyoxyethylene alkyl ethers and/or polyoxyethylene phenol ethers to a final hydrophobic lipophilic balance of approximately 13.5, as classically defined by Griffin, may be employed. (“Calculation of HLB Values of Non-Ionic Surfactants”, Journal of the Society of Cosmetic Chemists, 5 (4): 249–56; Okada M, Matsui H, Ito Y, Fujiwara A, Inano K., “Low-density lipoprotein cholesterol can be chemically measured: a new superior method”, J Lab Clin Med. 1998 Sep;132(3):195-201; Nauck M, Warnick GR, Rifai N.. “Methods for measurement of LDL-cholesterol: a critical assessment of direct measurement by homogeneous assays versus calculation”, Clin Chem. 2002 Feb;48(2):236-54.). An exemplary LDL-C protecting agent comprises 0.24% EMULGEN 66 (polyoxyethylene alkyl ether) and 0.6% EMULGEN 90 (polyoxyethylene distyrenated phenyl ether) commercially available from Kao Chemicals. Similar surfactants are available from other cosmetic and chemical manufacturing companies, as detergents and surfactants are composed of similar common hydrophobic and hydrophilic chemical building blocks. As such, equivalent detergents are available at many companies but with different trade names. Non-LDL and LDL cholesterol have different observable reactivities towards such detergents and surfactants (Sugiuchi H, Irie T, Uji Y, Ueno T, Chaen T, Uekama K, Okabe H., “Homogeneous assay for measuring low- density lipoprotein cholesterol in serum with triblock copolymer and alpha-cyclodextrin sulfate”, Clin Chem. 1998 Mar;44(3):522-31.) [0024] R2 also comprises a blend of cholesterol esterase and cholesterol oxidase which can be reconstituted to a final activity of 500 activity units/L to 10,000 activity units/L. These enzymes may be sourced from one of a variety of microorganism genres (e.g., Pseudomonas or Nocardia) or produced recombinantly. Cholesterol within non-LDL lipoproteins exists as free cholesterol molecules and as multiple species of cholesterol esters. Thus, cholesterol esterase is used to convert cholesterol esters to free cholesterol, where cholesterol oxidase can then convert free cholesterol molecules to hydrogen peroxide and cholestenone. [0025] In an aspect, R2 optionally comprise a peroxidase that converts the chemically- reactive hydrogen peroxide compound that is generated by cholesterol oxidase to an inert compound. One example is catalase at a final concentration of 10,000 – 500,000 activity units/L. [0026] R2 may further comprise a buffering chemical and a non-ionic salt, whose identity, concentrations and pH values, may conform to those as described for R1. [0027] An exemplary R2 is available commercially as a pre-formulated solution from Randox Laboratories Ltd, FUJIFILM Wako Pure Chemical Corporation, Beckman Coulter and other clinical diagnostics chemical distributers. R2 may be sold as part of an assembled kit or separately as an individual unit. [0028] For high-throughput processing, sample processing step 2 occurs in a multiwell plate (e.g., a 96 well plate) capable of holding volumes from 0.05 mL to 0.2 mL, e.g., typical plates compatible with commercial thermal cyclers, and uses calibrated pipettes or automated liquid handling systems for liquid transfers. Optional mixing to homogeneity occurs while samples are still maintained at 0°C to 10°C. Mixing can occur manually using a pipette by aspirating and dispensing the resulting solution multiple times until homogeneity is achieved. Mixing can also be performed using a shaker set at 250 to 3000 rpm for time periods of 10 seconds to 5 minutes. [0029] Once optional mixing is performed, samples may be incubated at an elevated temperature to activate the enzymes contained in R2 and permit them to destroy non-LDL cholesterol. The plate may be covered with a commercially available seal or a plastic cap-mat and incubated for 10 seconds to 10 minutes at a temperature that ranges from 20°C to 40°C until non-LDL cholesterol is destroyed. [0030] After the completion of the incubation, the reaction is optionally arrested until the extraction step described below is performed. A first method to arrest the reaction is to decrease the temperature to 0°C to 10°C, which kinetically prevents the enzymes in R2 from acting on the sample. A second means to arrest the reaction is to deactivate the enzymes by chemical means. Addition of an inhibitor or changing the solution pH to be acidic or basic, such that the enzymes are no longer active, serves this purpose. For example, addition of a solution of Fe³⁺ (e.g., Fe(III)Cl3), to a final concentration of 10 mM to 200 mM, inhibits the enzymes in the sample solution containing R2, if the said R2 constitutes a preformulated solution from FUJIFILM Wako. The volume of any additional liquid must be accounted for in the dilution factor 2 (DF2) by including the additional volume in the final dilution 2 volume. PROCESSING STEP 3: EXTRACTION OF THE LDL-C [0031] In the next processing step, an extraction, e.g., a two-phase extraction of the sample solution, is performed to extract the LDL-C. In the extraction, the remaining and intact cholesterol-containing macromolecules are denatured by an organic solution, thereby allowing release of cholesterol species into the organic phase. The organic solutions separate from the aqueous solutions, forming two layers or phases. Highly hydrophobic molecules, like cholesterol species, partition almost exclusively into the organic phase, whereas salts partition into the aqueous phase. This is helpful as salts are typically not compatible with mass spectrometry. Sampling of the organic phase is then performed in order to determine the LDL-C concentration using LC-tandem mass spectrometry. [0032] Two-phase extraction results in further dilution of the solution obtained from sample processing step 2. A volume of such solution, hereby abbreviated SV2, ranging from 5 μL to 100 μL, is added to reagent 3 (R3). Volumes of R3 may be in the range of 200 μL to 800 μL, so as long as the mixture of R3 and the sample results in two distinguishable layers with an identifiable organic layer above or below the aqueous phase. [0033] R3 comprises a first organic component and a second organic component. The first organic component is a water-immiscible organic liquid. Examples include hexane, heptane, nonane, diethyl ether, ethyl acetate, methyl tert-butyl ether and chloroform, preferably hexane. The volume percentage of this first component in R3 may range from 50% to 85% (v/v). [0034] The second organic component of R3 is an organic liquid that is miscible with both water and the first organic component of R3. Examples of the second organic component include methanol, isopropanol, and ethanol. This second organic component comprises approximately the remaining percent volume of R3 after subtracting that of the first component and thereby may be in a range from 15% to 50% (v/v). [0035] R3 also comprises internal standards of known concentrations that behave in a similar or identical manner as the corresponding analytes, but are distinguishable by the final instrumental analysis. One such internal standard, IS1, is detectably-labeled cholesterol ester, available from chemical supply companies, for example from Avanti Polar Lipids, Inc. or Cambridge Isotope Laboratories, Inc., that is packaged in liquid form or as a lyophilized powder. A second internal standard, IS2, is detectably-labeled free cholesterol, also available from specialized chemical supply companies. [0036] In an aspect, these internal standards are chemically identical to the analytes except one or more atoms have been replaced with a non-radioactive, stable isotope possessing a different atomic mass. As such the IS1 and IS2 behave identically as cholesterol esters and cholesterol in the described assay. Acceptable substitutions for carbon-12, oxygen-16 and hydrogen-1 in the cholesterol and cholesterol ester analytes are carbon-13, oxygen-18 or hydrogen-2 (deuterium), respectively. Isotopes within the internal standard are placed such that the mass detected in the instrumental analysis described below is greater than 1.8 units from that of its respective cholesterol or cholesterol ester analyte. [0037] Internal standards, IS1 and IS2, are reconstituted into R3 at an original concentration in molar units, [IS1]o and [IS2]o, to be in measurable range of the instrumental analysis. The additional sample dilution and the internal standard dilution that occurs during two-phase extraction, hereby termed dilution factor 3 (DF3), can be accounted for by taking the quotient of the reagent 3 volume divided by the added sample volume, SV2. ^^^^^^ ^^ ^3 (μ^) ^^^^^^^^ ^^^^^^ 3 ⁽^^3⁾ = ^^^^^^ ^^ ^^^^^^^ ^^^^^^ ^^^^ ^^^^^^^^^^ ^^^^ 2 (^^2) ( μ^)

plate (e.g., a 96 well plate) capable of holding volumes from 0.6 mL to 2.0 mL, using calibrated multi-channel pipettes or automated liquid handling systems for liquid transfers. Mixing to equilibration is preferred to complete the two-phase extraction, which permits the analytes and internal standards to partition into the water-immiscible organic phase. The plate may be covered with a commercially available seal or a plastic cap-mat and shaken for 0.25 minutes to 24 hours, at temperatures from 4°C to 40°C, at a rotation speed from 100 rotations per minute (rpm) to 3,000 rpm. Mixing can also occur by pipetting. The two phases will separate automatically after maintaining the plate without agitation but can be hastened by a brief centrifuge spin at 100 – 2,000 x g for 5 seconds or greater. [0039] After extraction, an LDL-C sample may be removed from the water-immiscible organic layer of each sample and deposited into a fresh vessel that may be a multiwell plate capable of holding volumes of 0.05 mL to 2.0 mL. The sample may be directly used in the instrumental analysis below. Furthermore, the sample may be evaporated to dryness, for example by using a commercial evaporator, and reconstituted in another solvent compatible with the instrumental analysis. LDL-C DETECTION AND MEASUREMENT [0040] Cholesterol in LDL consists of both free cholesterol, represented herein by FC, and by multiple species of cholesterol esters, represented herein in aggregate by CE. These analytes are measured using LC-tandem MS, wherein CE and FC are eluted from an analytical column at distinct times, wherein they are ionized using an atmospheric pressure chemical ionization source that is used to introduce the analytes into the MS, and wherein all species of FCs and CEs are detected at a primary mass-to-charge ratio (m/z) of approximately 369.4. [0041] Samples may be loaded into the LC auto-sampler and 0.5 μL to 50 μL of sample is injected into a stream of mobile phase A. Injection volumes are selected to be within the accurate volume sampling range of the LC instrument and also such that a generated cholesterol signals are within the linear measurement range of the MS instrumentation. [0042] An LC column is added in-line with the mobile phase flow path and is chosen such that FC and CE elute off the column at two separate times during analysis. Hydrophilic interaction liquid chromatography (HILIC) columns are one example of a suitable analytical separation column. Upon injection of sample, separation of CE and FC may occur using only single mobile phase A, i.e., isocratically, where each analyte is slowed at a different rate by the column matrix in a consistent and predictable manner. Alternatively, the mobile phase may be altered gradually over time by mixing a separate solvent, mobile phase B, into mobile phase A. In such a gradient elution, one or both analytes bind to the column matrix under initial mobile phase conditions and require a change in mobile phase composition to be eluted. Examples of mobile phase A for use with HILIC columns include hexane, nonane or heptane. Examples of mobile phase B include more hydrophilic solvents ethanol, butyl alcohol, or isopropanol, and may be blended to a defined percentage (v/v) with mobile phase A solvents in order to form the final mobile phase B composition. Mobile phase B constituents must be miscible with mobile phase A. [0043] During a single sample analysis that maintains or changes solvent percentages over a programmed amount of time, the mobile phase stream and the cholesterol compounds contained therein are introduced into the tandem MS instrumentation using an atmospheric pressure chemical ionization (APCI) source. The source conditions may be tuned such that the analyte cholesterol compounds in the mobile phase are transformed into a gaseous state, where each analyte molecule contains a single positive ionic charge, exists in the vapor as a single molecule, and is broken down into a predominantly homogeneous population of molecules containing an m/z ratio of 369.4. That is, in the first MS detection cell, all both free cholesterol and all cholesterol species should possess a m/z ratio of approximately 369.4. The cholesterol ions with a m/z of 369.4 are filtered, such that they are directed into new cell within the MS and are collided with an insert gas (e.g., helium or nitrogen) in order to break down these molecules down into smaller fragments with a characteristic mass. The smaller, fragment ions are isolated by mass in a third MS cell and their signal is measured. The signal generated by one or more of these signature fragment masses is denoted by a transition from the original or parent m/z value (e.g., 369.4) to another m/z value of the measured fragment, which is recorded over time for both the CE and FC analytes. Internal standard signals are recorded in an identical manner, using a different transition that is unique to the standard and excludes signal from the analyte. Total 0107668.0116 P220319WO01 signal of each cholesterol analyte and standard is determined, for example, using software that is either packaged with the LC-tandem MS system or using stand-alone software. [0044] Care may be taken to ensure that all analytes and standards signals are measured in the linear response ranges of the MS instrumentation, which is established by routine and standard method validation procedures necessary for use of any clinical method. Instrumental analysis is set up such that measurements avoid bias due to interferences from other substances in the samples. In the disclosed method, the response of each standard was identical to the response of the corresponding analyte, i.e., the relative response factor for each analyte standard pair was equal to 1, which is often assumed in MS instrumental analysis but can and should be verified experimentally. CALCULATION OF THE SAMPLE LDL-C CONCENTRATION [0045] Cholesterol in lipoproteins consists of both free cholesterol (FC), and by multiple species of cholesterol esters (CE). The total LDL-C concentration in the sample is thereby calculated by the following equation. ^^^ − ! = ^^^(!") + ^^^(^!) wherein: 1) LDL(CE)is the measured concentration of all cholesterol esters in a particular sample 2) LDL(FC) is the measured concentration of free cholesterol in that sample. [0046] The measured concentration of free cholesterol is calculated by the following equation: $ ^ = _%&' 1 ^^ ₍₎₍ ∗ - ∗ ^^1 ∗ ^^2 ∗ ∗ 386.65 ∗ 100 ∗ 6^

wherein: 1) A FC-LDL is the area of the signal generated by the free cholesterol in the sample LDL particles. 0107668.0116 P220319WO01 2) A _FC-IS is the area of the signal generated by the free cholesterol internal standard. 3) [IS2]o is the original concentration of the free cholesterol internal standard in moles (mol) per liter (L). 4) DF1, DF2, and DF3 are dilutions factors defined in the description above in processing step 1, processing step 2, and the two-phase extraction, respectively. 5) 386.65 is the average formula weight of cholesterol in grams (g) per mole (mol). 6) 100 is a factor to convert the unit grams (g) per liter (L) to the unit milligrams (mg) per deciliter (dL), which is standard unit for cholesterol measurements in the United States of America. 7) PR is the fractional percent recovery of the measured serum or plasma or blood product sample. When the blood sample is a serum or plasma sample, PR is typically equivalent to 1.0. When the blood sample is a dried blood spot sample, the value of PR may be determined as explained below. [0047] The measured concentration of CE is calculated by the following equation: $ 1 ^^^⁽!"⁾ = _&7'()( ∗ -.^1 ∗ ^^1 ∗ ^^2 ∗ ^^3 ∗ 386.65 ∗ 100 ∗ $_&7'*+ ^/ ₀ 6^ wherein: 1) A CE-LDL is the area of the signal generated by the cholesterol esters in the sample LDL particles. 2) A _CE-IS is the area of the signal generated by the cholesterol internal standard. 3) [IS1]_o is the original total concentration of the cholesterol ester internal standard in moles (mol) per liter (L). 4) DF1, DF2, and DF3 are dilutions factors defined in the description above in processing step 1, processing step 2, and the two-phase extraction, respectively. 5) 386.65 is the average formula weight of cholesterol in grams (g) per mole (mol). 0107668.0116 P220319WO01 6) 100 is a factor to convert the unit grams (g) per liter (L) to the unit milligrams (mg) per deciliter (dL), which is the standard unit of cholesterol measurement in the United States of America. 7) PR is the fractional percent recovery of the measured serum or plasma or blood product sample. When the blood sample is a serum or plasma sample, PR is typically equivalent to 1.0. When the blood sample is a dried blood spot sample, the value of R may be determined. DETERMINATION OF FRACTIONAL PERCENT RECOVERY FACTOR, PR, IN DRIED BLOOD SPOTS [0048] Fractional percent recovery is a value between 0 and 1 that describes the fraction of serum recovered from a sample collected by the disclosed process, as compared to an established method. Fractional percent recovery for a venous blood sample is 1. Fractional percent recovery for dried blood spot samples is expected to be 0.25 – 0.85. The fractional percent recovery factor, PR, may be determined by comparing patient serum LDL-C values obtained by an established method to those matching samples obtained using the disclosed method. To calculate LDL-C for the purpose of determining PR, the modified equations below may be used, where recovery is not taken into account and where equation variables are defined in an identical manner as above. ^^^ − ! = ^^^(!") + ^^^(^!) $ ^^^(!") = _&7'()( ∗ -.^1/₀ ∗ ^^1 ∗ ^^2 ∗ ^^3 ∗ 386.65 ∗ 100

$ ^^^(^!) = _%&'()( ∗ -.^2/ ∗ ^^1 ∗ ^^2 ∗ ^^3 ∗ 386.65 ∗ 100 $ _{0 %&'*+} [0049] The measured LDL-C values calculated with the equations above are plotted against the corresponding values obtained via the established comparison method with the matched samples. With the calculated values plotted on the y-axis, a least squares regression analysis is performed. A correlation coefficient equal to or greater than 0.90 is expected, and the difference between the linear model and the experimental values should be equally and randomly distributed across the LDL-C measurement range, indicating that the recovery factor is the same in all sample ranges. The value of PR is equivalent to the slope of the least squares regression line. PATIENT POPULATIONS [0050] In an aspect, the methods described herein are particularly useful to quantitate LDL-C in a blood sample from a human subject age 2-11 years, specifically 9-11 years. Determination of LDL-C at ages 9-11 could lead to earlier intervention with statins, for example, reducing the risk of a life-threatening cardiovascular later during adulthood. [0051] In a specific aspect, the human subject aged 2 to 11 years, specifically 9-11 years has a family history of premature cardiovascular disease, or a parent with a familial hypercholesterolemia (FH) mutation in APOB, PCSK9, or LDLRAP1. [0052] In an aspect, the human subject is diagnosed with FH when the total LDL-C is greater than 135 mg/dl, specifically greater than 160 mg/dl. In an aspect, when the human subject is diagnosed with FH and the method may further comprise administering a therapeutically effective dose of a statin or a selective cholesterol absorption inhibitor to the child, and/or to siblings of the child. [0053] In another aspect, the subject is an adult subject such as a subject at risk of coronary artery disease (CAD). Subjects at risk of CAD include smokers, sedentary individuals, individuals with a poor diet, obese individuals, diabetics, individuals with high-blood pressure, and combinations thereof. In an aspect, the human subject is diagnosed with CAD when the total LDL-C is greater than 135 mg/dl, specifically greater than 160 mg/dl. [0054] The invention is further described by the following non-limiting examples. EXAMPLES EXAMPLE 1: EXTRACTION AND QUANTITATION OF LDL-C FROM SERUM SAMPLES [0055] Proficiency testing serum samples were obtained from the Center for Disease Control and Prevention (CDC). Using the disclosed process, the LDL-C levels were determined in ten samples chosen to represent the range of measurement values in a typical patient population. These determined levels were compared to the average values obtained by over 500 national labs that performed direct LDL-C measurements using the ROCHE COBAS C clinical analyzer and packaged reagents to make these measurements. [0056] Serum sample quantities of 4 μL were added to 156 μL R1, as defined in Table 1, in a 96 well plate and shaken at 800 rpm at ambient temperature for 2 hours. Dilution factor 1 (DF1) was thereby equivalent to 40. TABLE 1: COMPOSITION OF R1 Component Compound Concentration pH (if applicable) ′ ″

[0057] A quantity of 5 μL of the resulting diluted samples was removed and added to 95 μL of R2 contained in a 96 well plate that can be inserted into a thermal cycler for subjecting the samples to controlled temperature cycling. To maintain R2 and the resulting mixture at 0°C, the plate was positioned in an aluminum block that was immersed in an ice bath. All liquid transfers were performed using a 12-channel 200 μL pipette. Homogeneity was achieved by four rounds of pipette mixing. R2 was obtained from FUJIFILM Medical Systems, U.S.A., Inc. as product identification number, 993-00404 (L-Type LDL-C Reagent 1). Dilution Factor 2 was 20. To destroy the non-LDL-C, the plate was moved to an Applied Biosystems Veriti^TM thermal cycler model 9902 preset at 4°C. Samples temperatures were increased to 37°C for 50 seconds and then returned to 4°C. [0058] Two-phase extraction was performed by removing 70 μL of the resulting samples, adding the sample volume to 350 μL R3, as defined in Table 3, contained in a deep-well plate, and mixing by pipette for a total of 15 rounds of aspiration / dispersion. The multiwell plate was agitated for 30 s at room temperature at 1100 rpm and then centrifuged at 750 x g for 30 seconds. TABLE 2: COMPOSITION OF R3 Component Compound Concentration

2 2-propanol 40% (v/v) 3 Deuterated cholesterol 4 x 10^-8 molar

Polar Lipids, Inc, in a quantitated and concentrated liquid stock solution as a production identification numbers LM4100 and 330822L. Dilution factor 3 (DF3) was therefore equivalent to 5. [0060] For sampling and reconstitution, 75 μL of the resulting upper phase organic layer was removed. A molded plastic spacer, 16.5 mm in thickness that contained holes for 96 pipette tips positioned to be in line with each well of the multi-well plate, was placed on top of the sample plate and used remove sample from a uniform depth in the final two-phase extraction solution such that only the upper organic phase was withdrawn. The removed samples were transferred to a new 96-well plate. The solvent was evaporated by incubation in an Apricot Designs^TM EVX192 evaporator for 5 minutes under a stream of 40°C N2 (g). Sample was reconstituted in 75 µL hexanes. [0061] Samples were analyzed using a Sciex API 4500 Triple Quad^TM mass spectrometer connected to a Shimadzu Nexera X2 liquid chromatography system. Cholesterol species and standards were fractionated over a 50 mm x 2.1 mm Kinetex^TM 1.7 μm HILIC column (Phenomenex) (part number 00B-4474-AN) by ramping the solvent from 100% hexanes (mobile phase A) to 2.5% ethanol in hexanes over 2 minutes, utilizing a mobile phase B constituted of 25% ethanol in hexanes. Data was acquired using an APCI source by collecting multiple reaction monitoring (MRM) scan data over the course of each sample run for the following m/z ratio transitions: 369.4 (m/z) ^ 147.1 (m/z), 376.4 (m/z) ^ 147.1 (m/z), and 385.4 (m/z) ^ 109.1 (m/z). These represented specific signals for cholesterol and cholesterol esters, the internal standard cholesterol and cholesterol esters, and the cholestenone produced by the destruction of non-LDL-C. Cholesterol esters ran through the column without binding, whereas cholesterol was release from column near the end of the gradient, thereby separating free cholesterol and cholesterol ester compounds. Additional instrumental analysis parameters are listed in Table 3. 0107668.0116 P220319WO01 TABLE 3: INSTRUMENT PARAMETERS Parameter Value Flow rate 06 mL/min [0

062] Data was processed with MultiQuant 3.0.3 software packaged with the LC- tandem MS instrumentation. Cholesterol ester analytes and the corresponding standard signals were observed at a retention time of 15 seconds, whereas free cholesterol and standard signals were observed at a retention time of 1.6 minutes. The signal, recorded in counts per second, for the two analytes and standards were integrated over time to produce an area measurement for each peak. Total LDL-C in mg/dL was calculated using the equations below. ^^^ − ! = ^^^(!") + ^^^(^!) $ 1 ^^^⁽!"⁾ = _&7'()( ∗ .^1 ∗ ^^1 ∗ ^^2 ∗ ^^3 ∗ 386.65 ∗ 100 ∗ $_&7'*+ ^{- /} ₀ 6^ $_%&'( 1 ^^^ = ₎₍ ∗ - ∗ ^^1 ∗ ^^2 ∗ ∗ 386.65 ∗ 100 ∗ 6^

wherein: 1) A_CE-LDL is the cholesterol ester area for the sample 2) ACE-IS is the cholesterol ester area for the internal standard 3) A_FC-LDL is the free cholesterol area for the sample 4) AFC-IS is the free cholesterol area internal standard 5) [IS1]o = 4 x 10^-8 M and is the cholesterol ester internal standard concentration 6) [IS2]_o = 4 x 10^-8 M and is the free cholesterol internal standard concentration 7) DF1 = 40 8) DF2 = 20 9) DF3 = 5 10) PR = 1.00 [0063] A total of four replicates for each sample was acquired over five days and the spread of the measures taken on each day was calculated in terms of intraday precision, found in the table below (Table 4) as percent coefficient of variation (%CV = (100 * standard deviation / average value)). The maximum %CV was 15.2%, though the median %CV was 5.8 %, within acceptable limits for a quantitative assay for a clinical method as established by clinical laboratory regulatory agencies.

TABLE 4: SERUM METHOD - INTRADAY PRECISION (%CV) Serum method - Intraday precision (%CV) 5 %

[0064] The average sample value of each day was similarly compared via calculation of the interday precision as a %CV, as displayed in the table below (Table 5). The maximum %CV was 6.8%, an acceptable value using the same metrics stated above.

TABLE 5: SERUM METHOD - INTERDAY PRECISION (%CV) Serum method - Interday precision (%CV) s

[0065] The average value of each day obtained by the disclosed process was also compared to the expected value as a percent difference (Table 6). The expected value was the average value obtained by the over 500 national labs that utilized the Roche Cobas clinical analyzer and packaged reagents to make the direct LDL-C measurements. As with the precision measurements, the accuracy was within acceptable limits for a clinical test, possessing a mean absolute difference of 3.7% from the expected value.

TABLE 6: SERUM METHOD – ACCURACY Serum method – Accuracy 5 % % % % % %

[0066] The average values obtained each day were also compared graphically to the expected values and a linear curve fit was calculated using standard least squares regression (Fig. 1). The correlation coefficient was 0.983, and the slope of the resulting line was 0.979 ± 0.026 (standard error) which indicates the disclosed process produced similar values as the reference method. EXAMPLE 2: EXTRACTION AND QUANTITATION OF LDL-C FROM DRIED BLOODS SPOTS [0067] Dried blood spot samples and corresponding serum samples were collected from 34 adult patients at the University of Wisconsin Hospitals and Clinics. LDL-C values were determined in the dried blood spots using the disclosed process for comparison to serum LDL-C values that were measured at UW Health Clinics. [0068] Using a Perkin Elmer Dried Blood Spot Puncher (model 1296-081), a single 1/8^th inch circular punch that contained 3.2 μL of blood was removed from each dried blood spots sample and deposited into a 96 well plate. A quantity of 100 μL of R1 was added to each sample 0107668.0116 P220319WO01 well, where R1 is identical to the formulation described in Example 1 above. The plate was sealed, centrifuged for 2 minutes at 750xg, transferred to a Perkin Elmer TriNEST^TM incubator shaker (model 1296-0050) and agitated for 2 hours at 37°C and 800 rpm. Assuming the samples contained 1.6 μL of serum, dilution factor 1 (DF1) was therefore equivalent to 62.5. [0069] A quantity of 7.5 μL of the resulting diluted samples was removed and added to 92.5 μL of R2 contained in a separate 96 well plate compatible with thermal cyclers. [0070] Processing step 2 and two-phase extraction then proceeded as described in example 1 above. Dilution Factor 2 (DF2) and dilution factor 3 (DF3) were therefore equivalent to 13.33 and 5, respectively. [0071] For sampling and reconstitution,100 μL of the resulting upper phase organic layer was removed and added to new multi-well plate in a similar manner as described in Example 1. The solvent was evaporated by incubation in an Apricot Designs^TM EVX192 evaporator for 5 minutes under a stream of 40°C N2 (g). Sample was reconstituted in 50 μL hexanes. [0072] LDL-C detection, measurement, and data processing were performed as described in Example 1 above. [0073] To determine the fractional percent recovery value, LDL-C values were calculated using the equations below. ^^^ − ! = ^^^(!") + ^^^(^!) $ ^^^(!") = _&7'()( ∗ -.^1/₀ ∗ ^^1 ∗ ^^2 ∗ ^^3 ∗ 386.65 ∗ 100 $_&7'*+ $ ^^^ = _%&'()( ∗ - ∗ ^^1 ∗ ^^2 ∗ ^^3 ∗ 386.65 ∗ 100

wherein: 1) ACE-LDL is the cholesterol ester area for the sample 2) A_CE-IS is the cholesterol ester area for the internal standard 3) A_FC-LDL is the free cholesterol area for the sample 4) AFC-IS is the free cholesterol area internal standard 5) [IS1]_o = 4 x 10^-8 M and is the cholesterol ester internal standard concentration 0107668.0116 P220319WO01 6) [IS2]_o = 4 x 10^-8 M and is the free cholesterol internal standard concentration 7) DF1 = 62.5 8) DF2 = 13.33 9) DF3 = 5 [0074] These calculated LDL-C values were compared to those obtained to those determine by UW Health clinics on the corresponding serum samples and plotted below in Fig. 2. A least squares regression was run on the set of matching values to yield a best fit line of y= 0.6913 x + 2.58. The correlation coefficient was 0.958 indicating a high level of correlation between the two methods. The differences between the observed values and the best fit line were equally and randomly distributed across the range of measured LDL-C values from approximately 36 mg/dL to 255 mg/dL. The slope of the line, 0.6913, was therefore a reliable estimate of the fractional percent recovery factor. [0075] Total LDL-C in mg/dL, taking the recovery into account, was therefore calculated using the equations below. ^^^ − ! = ^^^(!") + ^^^(^!) $_&7'()( 1 ^^^(!") = ∗ -.^1/₀ ∗ ^^1 ∗ ^^2 ∗ ^^3 ∗ 386.65 ∗ 100 ∗

$ ( ⁾ _%&'() 1 ^^^ ^! = ₍ ∗ .^2 ∗ ^^1 ∗ ^^2 ∗ ^^3 ∗ 386.65 ∗ 100 ∗ $_%&'*+ ^{- /} ₀ 6^ where variables are as defined in the above equations and PR = 0.6913 [0076] The dried blood spots samples values obtained by the disclosed process were compared to those determine by UW Health clinics on the corresponding serum samples and were graph below in Fig. 3. The correlation coefficient was 0.958, and the slope of the resulting line was 0.999 ± 0.053 (standard error), thereby indicating the disclosed process produced similar values as the reference method after adjusted for serum recovery from the dried blood spot samples. [0077] The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. [0078] While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS 1. A method of extracting and quantitating low density lipoprotein cholesterol (LDL-C) in a blood sample, comprising diluting or rehydrating the blood sample in an aqueous reagent 1 (R1) to provide a diluted sample having a first dilution factor (DF1); incubating a volume (SV1) of the diluted sample with an aqueous reagent 2 (R2) to provide a reaction solution having a second dilution factor (DF2), wherein incubating is for a time and temperature to degrade non-LDL cholesterol while maintaining LDL-C to provide a reacted sample comprising the LDL-C, wherein R2 comprises an LDL-C protecting agent, a cholesterol esterase enzyme, a cholesterol oxidase enzyme, and optionally a peroxidase; optionally arresting the reaction in the reacted sample to inhibit further cholesterol oxidase enzyme activity; extracting the LDL-C from the reacted sample by mixing a volume of the reacted sample (SV2) and a reagent 3 (R3) and removing a water-immiscible organic liquid phase sample comprising the LDL-C and an internal standard (IS), wherein R3 comprises the water-immiscible organic liquid, an organic liquid miscible with both water and the water-immiscible organic liquid, and the IS, wherein the IS comprises isotope-labeled cholesterol ester (IS1) and isotope- labeled free cholesterol (IS2), and wherein the water-immiscible organic liquid phase sample has a third dilution factor (DF3); optionally drying a portion of the water-immiscible organic liquid phase sample and reconstituting it in a mobile phase to provide a chromatography sample; separating cholesterol esters (CE) and free cholesterol (FC) in the water-immiscible organic liquid phase sample or the chromatography sample and determining areas of sample LDL-free cholesterol (A FC-LDL), sample LDL cholesterol esters (A CE-LDL), free cholesterol internal standard (A FC-IS) and cholesterol esters internal standard (A CE-IS) by liquid chromatography-tandem mass spectrometry (LC-MS/MS); and quantitating total LDL-C as the sum of A FC-LDL and A CE-LDL in the sample normalized by A FC-IS and A CE-IS.

0107668.0116 P220319WO01 2. The method of claim 1, wherein the LDL-C is calculated by the following equation: LDL-C (mg/dL) = LDL-CE (mg/dL) + LDL-FC (mg/dL) = $ 1 ^^^⁽^!⁾ = _%&'()( ∗ .^2 ∗ ^^1 ∗ ^^2 ∗ ^^3 ∗ 386.65 ∗ 100 ∗ $ _*+ ^{- /} _{0 %&'} 6^ + $ ^ !" = _&7' 1 ^^ ( ) ₍₎₍ ∗ -.^1/₀ ∗ ^^1 ∗ ^^2 ∗ ^^3 ∗ 386.65 ∗ 100 ∗ 6^

wherein [IS2]o is the original concentration of the free cholesterol internal standard in moles (mol) per liter (L); [IS1]_o is the original total concentration of the cholesterol ester internal standard in moles (mol) per liter (L); 386.65 is the average formula weight of cholesterol in grams (g) per mole (mol); PR is the fractional percent recovery of the blood sample; and 100 is a factor to convert the unit grams (g) per liter (L) to the unit milligrams (mg) per deciliter (dL). 3. The method of claim 1 or 2, wherein the sample is a dried blood spot sample (DBS). 4. The method of claim 3, wherein the DBS is a 3.2 mm sample equivalent to 3.2 μL of whole blood. 5. The method of claim 2, wherein the sample is a DBS and PR is 0.25 – 0.85. 6. The method of claim 1, wherein the sample is a venous blood sample. 7. The method of claim 2, wherein the sample is a venous blood sample, and R is 1. 8. The method of claim 1, wherein R1 comprises 5 – 200 mM buffer, 10 mM – 400 mM ionic salt, and 0.5 mM – 50 mM reducing agent to provide a pH of 6.0 to 8.0.

9. The method of claim 1, wherein the LDL-C protecting agent of R2 comprises a mixture of polyoxyethylene alkyl ether and polyoxyethylene distyrenated phenyl ether, and the cholesterol esterase enzyme and the cholesterol oxidase enzyme are from Pseudomonas or Nocardia. 10. The method of claim 1, wherein the reaction in the reacted sample is arrested by adding a solution of Fe³⁺, or decreasing the temperature. 11. The method of claim 1, wherein the water-immiscible organic liquid is hexane, heptane, nonane, diethyl ether, ethyl acetate, methyl tert-butyl ether, or chloroform; and the water-miscible organic liquid is methanol, isopropanol, or ethanol. 12. The method of any of the foregoing claims, wherein the LDL-C ranges from 36 mg/dL to 255 mg/dL. 13. The method of any of the foregoing claims, wherein the assay is a high throughput assay. 14. The method of any of the foregoing claims, wherein the blood sample is from a human subject age 2-11 years, specifically 9-11 years. 15. The method of claim 14, wherein the human subject aged 2 to 11 years. specifically 9-11 years, has a family history of premature cardiovascular disease, or a parent with a familial hypercholesterolemia (FH) mutation in APOB, PCSK9, or LDLRAP1. 16. The method of claim 14 or 15, wherein the human subject is diagnosed with FH when the total LDL-C is greater than 135 mg/dl, specifically greater than 160 mg/dl.

17. The method of claim 16, wherein the human subject is diagnosed with FH and the method further comprises administering a therapeutically effective dose of a statin or a selective cholesterol absorption inhibitor to the child, and/or to siblings of the child. 18. The method of any of claims 1-13, wherein the subject is an adult subject. 19. The method of claim 18, wherein the adult subject is at risk of coronary artery disease (CAD), wherein the adult subject is a smoker, a sedentary individual, an individual with a poor diet, an obese individual, a diabetic, and individual with high-blood pressure, or a combination thereof. 20. The method of claim 19, wherein the human subject is diagnosed with CAD when the total LDL-C is greater than 135 mg/dl, specifically greater than 160 mg/dl.