Light Scattering Detection Method Research Articles

Anti-osteoporosis effects of Anemarrhenae Rhizoma / Phellodendri Chinensis Cortex herb pair and its major active components in diabetic rats and zebrafish

Ethnopharmacological relevanceAnemarrhenae Rhizoma/Phellodendri Chinensis Cortex (AR/PCC) herb pair has been widely used in traditional Chinese medicines for the treatment of diabetic osteoporosis. However, the anti-diabetic osteoporotic active components of AR/PCC remain unclear. This study aimed to explore the major active ingredients in AR/PCC for its protective effects against bone deterioration induced by diabetes. Materials and methodsThe aqueous extracts of AR/PCC with different proportions (AR:PCC = 1:3, 1:2, 1:1, 2:1 and 3:1, w/w) were prepared. Streptozotocin-induced diabetic rats were orally administrated with the AR/PCC extracts. The absorbed phytochemical compounds in serum of diabetic rats were identified by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry method and their contents in the AR/PCC extracts were determined by high performance liquid chromatography-ultraviolet detector-evaporative light scattering detector method. The absorbed compounds in the extracts were considered as the major potential active components in AR/PCC, and their combination was defined as M-AR/PCC. A component-knockout approach was applied to evaluate the contribution of each compound in M-AR/PCC. The larvae and adults of diabetic zebrafish models were then used to evaluated the anti-diabetic osteoporotic performance of the M-AR/PCC. The real-time reverse transcription polymerase chain reaction technique was applied to study the regulation effects of M-AR/PCC on osteogenesis and osteoclastgensis in diabetic zebrafish models. ResultsThe phenotypes of diabetic osteoporosis rats induced by streptozotocin were reversed by the oral administration of AR/PCC extracts with different ratios, as evidenced by the increased bone mineral density, bone volume density, trabecular thickness, trabecular number, and decreased trabecular separation of femoral metaphysis. Seven phytochemical compounds were detected in the serum and their contents in AR/PCC varied dramatically with different proportions, including 1 xanthone glycoside and 6 alkaloids. By using diabetic zebrafish larvae model and compound-knockout strategy, each compound in M-AR/PCC were proved to play an indispensable role in the positive regulatory actions in the bone mass of diabetic zebrafish. Furthermore, the herb pair with a ratio of 1:1 and the related M-AR/PCC showed the best therapeutic effects on diabetic osteoporosis. They showed similar performances on the inhibition of the tartrate-resistant acid phosphatase activity and the promotion of the alkaline phosphatase activity in diabetic adult zebrafish model. The M-AR/PCC treatment could decrease the blood glucose, upregulate the mRNA expression levels of osteoblast-related genes (alp, runx2b and opg) and downregulate the expression of osteoclast-related genes (acp5α, rankl and sost) in streptozotocin-induced zebrafish. ConclusionAR/PCC herb pair and its major active components possess potent anti-diabetic osteoporotic effect on streptozotocin-induced in vivo models. The combination of the seven active compounds derived from AR/PCC herbal pair could be a potential agent for protection against osteoporosis associated with diabetes.

Beeswax Fractions Used as Potential Oil Gelling Agents

AbstractThe objective of this work was the fractionation of beeswax to investigate the phase behavior of binary blends of the fractions and their potential use as gelling agents in edible oleogels. We have extracted seven distinctive fractions, using preparative flash chromatography with eluents permitted to be used in the processing of food raw materials. The odor profile of the fractions was characterized. The high purity of collected fractions was shown through thin‐layer chromatography, Fourier‐transform infrared spectroscopy, and high‐performance liquid chromatography with an evaporative light scattering detector methods for hydrocarbons (94.8%), monoesters (97.6%), and mixed mono‐, di‐, and triesters fraction (98.7%). Free fatty acids and fatty alcohol fractions had lower purity which equals 82.6% and 39%, respectively. The analysis of the binary pseudo phase diagram revealed that all fractions combined with hydrocarbons express eutectic behavior. Combinations of all other fractions resulted in the formation of solid solutions. This study shows that the texture of oleogels can be improved by using a combination of various fractions of beeswax instead of native wax.

Erythrocyte Membrane and Plasma Fatty Acids Composition in Patients with Coronary Heart Disease Requiring Percutaneous Angioplasty

Aims: Atherosclerosis is associated with oxidative stress and fatty acid composition plays a critical role in vascular endothelial dysfunction and injury leading to the coronary atherosclerotic progression. However, the correlation between the fatty acid profile and coronary atherosclerosis is debatable. The goal of this study is to assess the erythrocyte membrane and plasma fatty acid composition in patients with coronary heart disease.
 Methods: The erythrocyte membrane and plasma distributions of fatty acids were quantified in patients with coronary heart disease (n = 30, group A) which needs both intensive medication and elective percutaneous coronary intervention and age-matched controls (n = 38, group B) using high-performance liquid chromatography combined with evaporative light scattering detection method. Baseline data were extracted from medical records.
 Results: Logistic regression analysis demonstrated that hypoalbuminemia (p = 0.010) and HbA1c (p = 0.005) are associated with required percutaneous coronary intervention. Although appropriate logistic regression model for percutaneous coronary intervention could not be obtained by incorporating fatty acid components, percutaneous coronary intervention was correlated mostly to the increased oleic acid and decreased stearic acid in both erythrocyte membrane and plasma in receiver-operating characteristic analysis.
 Conclusion: This single-center, cross-sectional study indicated that erythrocyte membrane and plasma fatty acids have a potential impact on the coronary atherosclerotic progression which requires coronary intervention. Longitudinal studies are necessary to clarify the clinical role of fatty acids distribution as a novel atherogenic marker.

Simultaneous Identification of Characteristic Components in HPLC-PDA-ELSD Fingerprint Profile of Ginkgo biloba Leaves Extract

This is an updated fingerprint profile for the simultaneous identification of 29 compounds, mainly including characteristic -terpene trilactones and flavonoid glycosides from Ginkgo biloba leaves (EGb) by employing a high-performance liquid chromatography-photodiode array-evaporative light scattering detector method. Compounds 1 and 6 had not been previously either detected or described in any EGb samples. In general, these 29 compounds were distributed into 5 regions in the fingerprint according to their different structural properties. This efficient analytical method could be generally applied to the quality control of EGb761 and other commercially available EGb products in the Chinese active pharmaceutical ingredients market.

Comparative Analysis of Carbohydrates, Nucleosides and Amino Acids in Different Parts of Trichosanthes kirilowii Maxim. by (Ultra) High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry and Evaporative Light Scattering Detector Methods.

Trichosanthes kirilowii Maxim. is one of the original plants for traditional Chinese medicines Trichosanthis Fructus, Trichosanthis Semen, Trichosanthis Pericarpium and Trichosanthis Radix. Amino acids, nucleosides and carbohydrates are usually considered to have nutritional value and health-care efficacy. In this study, methods involving high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD), UV-visible spectrophotometry and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) were established for quantifying carbohydrates (fructose, glucose, stachyose, raffinose and polysaccharide), fourteen nucleosides and twenty one amino acids. Moreover, sixty-three samples from nine different parts, including pericarp, seed, fruit pulp, stem, leaf, main root, main root bark, lateral root and lateral root bark of T. kirilowii from different cultivated varieties were examined. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. The results showed that the average content of total amino acids in roots (15.39 mg/g) and root barks (16.38 mg/g) were relatively higher than for others. Contents of nucleosides in all parts of T. kirilowii were below 1.5 mg/g. For carbohydrates, fruit pulp has a higher content than others for glucose (22.91%), fructose (20.63%) and polysaccharides (27.29%). By using partial least-squared discriminate analysis (PLS-DA), Variable importance in the projection (VIP) plots and analysis of variance (ANOVA) analysis, the characteristic components of the different organs (fruit, stems and leaves, roots) were found. This analysis suggested there were potential medicinal and nutritive health care values in various parts of the T. kirilowii, which provided valuable information for the development and utilization of T. kirilowii.

Dipalmitoylphosphatidylcholine (DPPC): Annealing Strategy to Mitigate Variability in Thermotropic and Moisture Sorption Behavior

Dipalmitoylphosphatidylcholine (DPPC) demonstrated complex differential scanning calorimetry (DSC) thermal behavior. Transitions below 100°C showed variability in their thermotropic reversibility. An experimental design employing a DSC heat-cool-heat-cool-heat cycle and modulated DSC were used to gain insight into the DPPC's complex thermal nature. An annealing strategy was developed to reduce DPPC's thermotropic variability, moisture uptake rate, and rate variability. Samples annealed at 110°C for 5 min provided a reproducible, thermally reversible material. The annealed material also exhibited an 8-fold decrease in moisture sorption rate and a statistically significant (p = 0.0233) 100-fold decrease in water sorption rate variability compared to DPPC “as is.” An optimized validated stability-indicating high performance liquid chromatography with evaporative light scattering detection method was developed and showed no change in DPPC chemical stability under the annealing treatment conditions.

Application of capability indices and control charts in the analytical method control strategy.

In this study, we assessed the usefulness of control charts in combination with the process capability indices, Cpm and Cpk , in the control strategy of an analytical method. The traditional X-chart and moving range chart were used to monitor the analytical method over a 2-year period. The results confirmed that the analytical method is in-control and stable. Different criteria were used to establish the specifications limits (i.e. analyst requirements) for fixed method performance (i.e. method requirements). If the specification limits and control limits are equal in breadth, the method can be considered "capable" (Cpm =1), but it does not satisfy the minimum method capability requirements proposed by Pearn and Shu (2003). Similar results were obtained using the Cpk index. The method capability was also assessed as a function of method performance for fixed analyst requirements. The results indicate that the method does not meet the requirements of the analytical target approach. A real-example data of a SEC with light-scattering detection method was used as a model whereas previously published data were used to illustrate the applicability of the proposed approach.

Stability analysis of tannin-based foams using multiple light-scattering measurements

Liquid foams were prepared by whipping aqueous solutions based on natural polyphenols, surfactant and crosslinker. The destabilisation mechanisms of such thick, opaque and viscous liquid foams were investigated for the first time with a light-scattering detection method using an optical analyser. The technique allowed separating processes such as drainage, coalescence and coarsening, and sedimentation, so that each of them could be investigated independently as a function of whipping time, surfactant amount, and temperature. The corresponding average bubble diameters could also be determined as a function of time. It was shown that this kind of optical analysis is quite relevant for optimising formulations and foaming conditions with the aim of recovering rigid foams after polymerisation, having a given homogeneous structure that might be useful for various applications such as thermal insulation or acoustic absorption.

Simultaneous Determination of Four Active Ingredients in Sargentodoxa cuneata by HPLC Coupled with Evaporative Light Scattering Detection.

A HPLC coupled with evaporative light scattering detection method had been developed for the simultaneous determination of 3,4-dihydroxyphenylethyl alcohol glycoside, salidroside, chlorogenic acid, and liriodendrin in the stem of Sargentodoxa cuneata. With a C18 column, the analysis was performed using acetonitrile and 0.2% formic acid aqueous solution as mobile phase in gradient program at a flow rate of 0.9 mL/min. The optimum drift tube temperature of evaporative light scattering detection was at 105°C with the air flow rate of 2.5 L/min. The calibration curves showed good linearity during the test ranges. This method was validated for limits of detection and quantification, precision, and reproducibility. The recoveries were within the range of 96.39%–104.64%. The relative standard deviations of intraday and interday precision were less than 2.90% and 3.30%, respectively. The developed method can be successfully used to quantify the four analytes in the stem of Sargentodoxa cuneata from various regions in China.

Contents Changes of Triterpenic Acids, Nucleosides, Nucleobases, and Saccharides in Jujube (Ziziphus jujuba) Fruit During the Drying and Steaming Process.

Chinese jujube (Ziziphus jujuba), a medicinal and edible plant, is widely consumed in Asian countries owing to the remarkable health activities of its fruits. To facilitate selection of the suitable processing method for jujube fruits, in this study their contents of triterpenic acids, nucleosides, nucleobases and saccharides after drying and steaming treatment were determined using ultra-high performance liquid chromatography and high performance liquid chromatography coupled with evaporative light scattering detector methods. The results showed that except for sucrose, the content levels of most analytes were increasing in the jujube fruits during drying treatment at 45 °C. The levels of cyclic nucleotides such as adenosine 3′,5′-cyclic monophosphate and guanosine 3′,5′-cyclic monophosphate, were significantly decreased after the fruits were steamed. Therefore, owing to the bioactivities of these components for human health, the dried fruits would be the better choice as medicinal material or functional food, and dried jujube fruit should not be further steamed.

Simultaneous determination of multiple platycosides with a single reference standard in Platycodi Radix by high-performance liquid chromatography coupled with evaporative light scattering detection.

A traditional external standard method using HPLC coupled with evaporative light scattering detection has been developed for fast and accurate determination of seven platycosides in Platycodi Radix. However, inevitable difficulties in reference standards preparation process, which are quite costly and time consuming, have made its application limited. To avoid this inconvenience, a simultaneous determination of multiple components with a single reference standard strategy, which could be realized by calibrating the standard curve with internal standard and response factors, was introduced to the HPLC coupled with evaporative light scattering detection method. This is the first time that an incorporation of these two methods has been realized. Among seven ingredients, platycodin D was selected as the internal standard for its relatively easy preparation and low cost. Moreover, according to the investigation on concentration-dependent effects over response factors and robustness test, platycoside E, deapioplatycodin D, platycodin D, and polygalacin D2 were chosen to be the indicators for this novel method. The present method has not shown statistically significant differences with a traditional external standard method as verified sample analysis by the F-test (p = 95%, n = 6).

Development and validation of a simple high performance liquid chromatography – evaporative light scattering detector method for direct quantification of native cyclodextrins in a cyclization medium

A simple HPLC–ELSD method was developed for the separation and quantification of native cyclodextrins. The technique was validated in the presence of two interfering matrices composed of byproducts from the cyclization medium. A fast separation of the compounds was achieved (in <20min) using a NUCLEODUR® C18 Pyramid column (150mm×4.6mm; particle size 5μm) at 30°C. The analytes were eluted using a linear gradient of acetonitrile and water containing 1% (v/v) of acetic acid at a flow rate of 0.3mL/min. Validation results showed that the method was accurate (93–110%) and selective. The precision was ≤5.7% for a hydrolyzed starch blank matrix spiked with cyclodextrins, and ≤6.2% for a blank matrix composed of a mixture of dextrin and glucose spiked with cyclodextrins. The limit of quantification was 0.05g/L for alpha- and 0.06g/L for beta- and gamma-cyclodextrins. The new HPLC–ELSD method could accurately quantify the three cyclodextrins directly in a cyclization medium, without pretreatment of the samples.

Analysis of L-Quebrachitol from the Waste Water of Rubber Latex Serum Using Hydrophilic Interaction Chromatography and Evaporative Light Scattering Detector Method

L-Quebrachitol (QCT) is a valuable active ingredient found in rubber latex serum. Due to its high polarity, quantitative analysis of QCT is difficult on traditional reverse phase liquid chromatography (RPLC). Hydrophilic interaction chromatography (HILIC) using a Shiseido PC HILIC column (250 × 4.6 mm) and an evaporative light-scattering detector (ELSD) was found to be convenient for the analysis of QCT. The mobile phase was acetonitrile–water (60:40, v/v), retention factor is 0.92. Outstanding detection linearity (5 × 10−5–4 g/L) and limit of detection (LOD, 8 × 10−6 g/L) was achieved. Using this method, 2.2% wt QCT was determined to be present in rubber latex waste water. The retention of QCT on PC HILIC, which was immobilized with phosphorylcholine, was also investigated theoretically, the interaction energy between QCT and phosphorylcholine group is −51.498 kJ/mol, and ΔH0 and ΔS0 for the retention of QCT on PC HILIC is −0.699 kJ/mol and 2.72 J/mol · K, respectively.

Capability measurement of size-exclusion chromatography with a light-scattering detection method in a stability study of bevacizumab using the process capability indices

In this study, we investigated if the size-exclusion chromatography coupled with light-scattering and refractive index detection (SEC/LS/RI) method is fitted for its intended purpose and checked if the analytical method is able to provide enough conforming results. For this, the process capability indices Cp, Cpk, and Cpm were computed. The traditional X-chart and moving range (MR) chart were used by the same analyst to monitor the equipment in the laboratory over a 1-year period. For this, a bovine serum albumin (BSA) sample (0.3mgmL−1) with a nominal Mw of 66.4kDa was analyzed each working day. The results confirmed that the analytical method is in-control and stable. To determine whether the given process meets the present capability requirement and runs under the desired quality conditions, the Pearn and Shu (2003) method based on the lower confidence bound C on Cpm was used. The estimator Cpm was 1.81, and the lower confidence bound C was 1.40. We therefore conclude that the true value of the method capability Cpm is no less than 1.40 with a 95% level of confidence. This result indicates that the method is satisfactory and no stringent precision control is required. The usefulness of this method was applied in the characterization of bevacizumab commercial pharmaceutical preparations stored under different conditions that lead to aggregation. In this case, the computed Cpm index was 0.98 (0.70, 1.26), which indicates that the method does not comply with the specification limits and needs to be revised. The quality improvement effort should: (1) reduce the uncertainty in the absolute Mw determination; (2) either move the process mean closer to the target value or reduce the process variation, i.e. improve the method accuracy (μ–T) and precision (σ2). On this point, the Bayesian posterior distribution of the mean and standard deviation pointed out the need to control the precision but specially accuracy in order to reduce the overall uncertainty of analytical method and thus, the method is capable.

Identification and Determination of the Major Triterpenes in Rhizoma Alismatis by HPLC-Evaporative Light Scattering Detection and HPLC/Electrospray Ionization-MSn

A simple and reliable HPLC-evaporative light scattering detection method was established for the determination of seven triterpenes in Rhizoma Alismatis, a commonly used herbal medicine. HPLC coupled with electrospray ionization-MS was applied to identify the triterpenes. The positive ion mode was used in MS detection, and the fragmentation patterns of the analytes were proposed. The quantitative method was validated for linearity, sensitivity, precision, and accuracy. All calibration curves showed good linearity (r2 > 0.9943) within the test ranges. This method showed good reproducibility with intraday and interday variations of less than 3.39 and 5.20%, respectively. The mean recoveries ranged from 96.06 to 103.5%. The proposed method was successfully applied for the quantitative analysis of the triterpenes in samples from four different habitats. The results indicated great variation of the contents of these components among the samples, and the developed assay could be considered as a suitable quality evaluation method for Rhizoma Alismatis.

Profiling and quantifying polar lipids in milk by hydrophilic interaction liquid chromatography coupled with evaporative light-scattering and mass spectrometry detection

In this work, a high-performance liquid chromatography with evaporative light scattering detection method has been developed and applied for quantification of the polar content of the lipid fraction in milk samples of different origin. From a chromatographic stand-point, a 4.6-mm I.D. hydrophilic interaction liquid chromatography column was employed to attain a baseline separation of major phospholipid classes contained in the various milk samples tested. Quantitative analysis was performed by the external calibration method using reference material solutions in the 5-100 mg/L concentration range. Analytical recoveries ranging from 57 to 100%, and repeatability data lower than 8.04% were obtained on a skimmed cow's milk sample. The crude cow milk was the most abundant (0.04%) in phospholipids and donkey milk was the poorest (0.004%). Quantitative differences were determined in the phospholipid content of the milk samples tested. Finally, characterization of phospholipid profile and fatty acid composition of the different samples was carried out by an ion trap-time of flight mass spectrometer and gas chromatography coupled to flame ionization and mass spectrometry detection. A thorough screening of the polar lipid composition of milk samples of different origin is here outlined, for the first time.

HPLC-ELSD Determination of Kryptofix 2.2.2 in the Preparations of 2-Deoxy-2-[18F]fluoro-d-glucose and other Radiopharmaceuticals

A new and simple high-performance liquid chromatography with evaporative light scattering detector method for the determination of Kryptofix 2.2.2 (K-222) in the radiopharmaceuticals of 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) and 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) was developed. A C18 column was used and the mobile phase was 10 % (v/v) methanol and 90 % (v/v) water (0.1 % trifluoroacetic acid, v/v) at a flow rate of 0.2 mL min−1. The drift tube temperature was 40 °C. The pressure of nebulizing gas (N2) was 3.0 bar. The gain was 10. Good separation of K-222 from main related substances could be achieved. Excellent linearity (r 2 = 0.9995) was obtained over the range of 5–100 μg mL−1. The precision ranged from 0.68 to 5.16 % (RSD) and the accuracy ranged from −3.05 to 2.62 % (RE). The limit of detection was 2 μg mL−1. This method offers simple, rapid and quantitative detection of K-222, thus making it acceptable for routine determination.

Heterogeneous Components of Chitosans

The main objectives of the research were to compare the components of partially N-deacetylated chitins prepared identically from native chitin and a chitin regenerated from a heavily deacetylated chitosan. Additionally, to determine if any of the water-soluble components would serve as substrates in a study of a Chitinase isolated from soy bean hull. The brief heating of suspended chitins in 20% (w/w) NaOH resulted in similar degrees of N-deacetylation, the native chitin giving DAc 0.84 and the regenerated chitin DAc 0.79-0.72, with DAc indicating the proportion of glucosamine residues that are acetylated. Evidence for the nature of the hydrolysis of acetamido groups was provided by analyses of the water-soluble and -insoluble Smith degradation products. The water-soluble fraction derived from the native chitin comprised very small amounts of erythrityl N-acetyl glucosaminoside (GlcNAc1E), erythrityl N,N'-diacetyl chitobioside (GlcNAc2E), and erythrityl N,N',N''-triacetyl chitotrioside (GlcNAc3E), each identified by MALDI-TOF mass spectrometry of the butanoyl derivative. The water-insoluble products, as analyzed by light scattering detection method of their butanoyl esters and corrected for their composition, had a molecular weight (Mw) of 25 kDa, corresponding to about 120 N-acetyl glucosaminyl repeating residues (DPw), contrasting to that of 140 kDa with DPw of 680 for the parent chitin. Much of the decrease in the molecular weight of the polymer occurs by the loss of sugar residues by alkaline peeling at reducing terminals. For the regenerated chitin (DAc 1.0), prepared by N-reacetylation of a commercial chitosan (DAc 0.15), the resulting Smith products comprised erythritol and a series of N-acetyl glucosaminyl erythritol homologues of up to at least 39 N-acetyl glucosaminyl repeating residues, reflecting greater heterogeneity in the hydrolysis of acetamido groups along the polymer chain than what was seen for the native chitin. Of the water-soluble Smith products, GlcNAc5-7E were good substrates for chitinase isolated from soybean hull.

Detection of Ascorbic Acid Using the Plasmonic Resonance Light Scattering Method Based on Silver Nanoparticle Probe

A resonance light scattering detection method of ascorbic acid is reported based on the formation of silver nanoparticles(NPs).Ascorbic acid(vitamin C,VC) could reduce Ag(NH3) 2+ to form the silver nanoparticles,resulting in strong plasmon resonance light scattering(PRLS) signals characterized at 544 nm,which could be measured using a common spectrofluorometer.It is found that the PRLS intensities were proportional to the ascorbic acid concentrations in the range of 0.2～7.0 μmol/L with R2=0.9988 and detection limit(3σ/k) of 8.1×10-9 mol/L.The presence of a physically adsorbed layer of the product of the redox reaction on the surface of colloidal silver could prevent irreversible aggregation of silver NPs.The conditions of the reaction and the interference of the common ions are investigated.For the determination of ascorbic acid in VC tablets and beverage samples.The recovery was within the range of 94.5% ～ 97.0%.Results suggested that the method possessed easy of implementation,high sensitivity,and better recovery,and so on.

Quantification of Phospholipids in Infant Formula and Growing Up Milk by High-Performance Liquid Chromatography with Evaporative Light Scattering Detector

Phospholipids (PLs) are well known for their excellent emulsifier properties and more recently for their biological functions, such as cell signing, brain development, immune function, heart health, and cancer prevention, besides their physiological role in membrane composition. In dairy products, PLs represent 0.2-1% of milk fat. The milk PLs comprise phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SPH) as the major compounds; phosphatidylinositol and phosphatidylserine are minor PLs. A new generation of dairy products claiming PL family content, such as SPH, is being produced; therefore, a validated method for quantifying PL families in dairy products is needed. In this study, an HPLC-evaporative light scattering detector method to quantify the most abundant milk PL families, i.e., PC, PE, and SPH, in infant formula and growing up milk was developed and validated.